Double-stranded RNA methods and compositions for insecticides

Abstract

Disclosed herein are pesticidal compositions and methods for controlling plant pests. Pesticidal compositions and methods comprise recombinant RNA molecules for RNA interference mediated target gene inhibition in plant pests. Further provided are transgenic plants expressing recombinant RNA molecules useful for controlling plant pests, as well as plant cells and plant products obtained thereby.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 520,023, filed August 16, 2023, entitled “DOUBLE STRANDED RNA METHODS AND COMPOSITIONS FOR INSECTICIDE,” which is incorporated herein by reference in its entirety.

[0002] Merging of sequence lists This application contains a sequence list that has been submitted via the Patent Center in .XML format and is incorporated herein by reference in its entirety. The WIPO sequence list was created on August 13, 2024, and the XML copy is named 066803-818366_Sequence listing.xml and is 41 kilobytes in size. Technical Field

[0003] This disclosure relates to the field of RNA interference (RNAi)-mediated gene silencing in insect pests. Background Technology

[0004] Controlling insect pests on agriculturally important crops is crucial for maximizing crop yields. Insect pests affecting plants belonging to the Solanaceae family include many Coleoptera pests, particularly those of potatoes (Solanum tuberosum), tomatoes (Solanum lycopersicum), eggplants (Solanum melongena), peppers (Solanum capsicum), and plants in the genus *Solanum* (e.g., *Solanum aculeastrum*, *S. bulbocastanum*, *S. cardiophyllum*, *S. douglasii*, *S. dulcamara*, *S. lanceolatum*, *S. robustum*, and *S. triquetrum*). Despite progress in recent decades in developing more effective methods and compositions for using chemical pesticides to control insect infestations in plants, the development of resistance to these compounds by insect pests remains a significant problem. Biological control methods, such as RNA interference (RNAi)-based biopesticides, offer alternative and unique approaches to integrated pest management.

[0005] RNAi is a natural biological process found in most eukaryotes, serving to defend against viruses or playing a role in the regulation of messenger RNA (mRNA) stability and translation. In some insects, such as the Colorado potato beetle (CPB) and other Coleoptera species, the RNAi pathway can be triggered upon ingestion of exogenous double-stranded RNA, potentially providing this technology for pest control. Therefore, there is an unmet need to identify novel targets in the insect genome and develop RNAi-based insecticides to cause insect pest death, growth arrest, or sterility. Summary of the Invention

[0006] In some aspects, this disclosure covers insecticidal compositions comprising an insecticidally effective amount of a recombinant RNA molecule, said recombinant RNA molecule comprising a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity to a portion of a target gene of an insect infecting a plant, said target gene encoding an mRNA sequence selected from SEQ ID No: 1, 4, 6 and 8.

[0007] In some aspects, the disclosed insecticidal composition comprises a recombinant RNA molecule containing at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID NO: 3, 5 and 10. In some aspects, the recombinant RNA molecule is double-stranded RNA (dsRNA), microRNA (miRNA), small interfering RNA (siRNA), hairpin RNA (hpRNA), or piwi-interacting RNA (pi-RNA).

[0008] In some aspects, the disclosed insecticidal composition comprises a recombinant RNA molecule, which is a double-stranded RNA (dsRNA) containing an RNA chain having a sequence selected from SEQ ID NO: 3, 5, and 10. In some aspects, the dsRNA is at least 17 base pairs in length.

[0009] In some cases, the insect is an adult, or in a larval or nymphal stage. In some cases, the plant-infecting insect is a Coleopteran. In some cases, the plant-infecting insect is an insect from the genus *Leptinotarsa*. In some cases, the plant-infecting insect is the potato beetle *Leptinotarsadecemlineata* (Colorado potato beetle), and the target gene encodes an mRNA sequence containing the nucleotide sequence of SEQ ID No: 1. In some cases, the plant-infecting insect is the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence containing the nucleotide sequence of SEQ ID No: 4 or 6. In some cases, the plant-infecting insect is the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence containing the nucleotide sequence of SEQ ID No: 8. In some aspects, the plant-infecting insect is the potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID No: 3, 5, and 10. In other aspects, the plant-infecting insect is the potato beetle (Colorado potato beetle), and the recombinant RNA molecule is dsRNA comprising an RNA strand having a sequence selected from SEQ ID No: 3, 5, and 10.

[0010] In some aspects, the disclosed insecticidal composition further comprises at least one component selected from carriers, excipients, diluents, surfactants, organosilicones, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide pesticides, safeners, insect attractants, and insect growth regulators. In some aspects, the insecticidal composition further comprises at least one pesticide reagent. In some aspects, the pesticide reagent is selected from potato glycoprotein (patatin), phytohemagglutinin, phytoecdysone, and insecticidal proteins.

[0011] In some respects, the disclosed insecticidal compositions are selected from the following forms: solid, liquid, powder, suspension, emulsion, spray, encapsulating agent, microbeads, carrier microparticles, film, solid matrix, soil irrigation agent, insect food, insect bait and seed treatment agent.

[0012] This article further discloses plants or seeds thereof treated with the disclosed insecticidal composition, wherein the plants exhibit improved resistance to insects.

[0013] In some aspects, this disclosure further covers recombinant DNA constructs comprising a heterologous promoter operatively linked to DNA encoding an RNA transcript comprising a sequence having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID NO: 3, 5, and 10. In some aspects, the recombinant DNA constructs wherein the heterologous promoter is functional for the expression of the RNA transcript in bacteria or fungi.

[0014] In some respects, heterologous promoters are functional in plant cells. This document further discloses recombinant vectors comprising the disclosed recombinant DNA construct. In further respects, this document provides plant chromosomes or plastids comprising the disclosed recombinant DNA construct. In some respects, it further provides transgenic plant cells having the disclosed recombinant DNA construct in their genome, transgenic plants comprising transgenic plant cells, or crop products produced by transgenic plants. In some respects, it provides seeds or fertile plant parts of the transgenic progeny of the disclosed transgenic plants.

[0015] In a further aspect, this disclosure covers a method for controlling plant infection by infective insects, the method comprising contacting the insect with dsRNA, said dsRNA comprising at least one portion having 17 or more adjacent nucleotides having a sequence having about 95% to about 100% complementarity to a target gene of the insect or a portion thereof, said target gene encoding an mRNA sequence selected from SEQ ID NO: 1, 4, 6, and 8. In some aspects of the method, the insect is an adult, or in a larval or nymphal stage. In some aspects, the plant-infective insect is a Coleoptera insect. In some aspects, the plant-infective insect is an insect from the genus *Lepidocybe*. In some aspects, the insect is the potato beetle (*Cotinus potato beetle*).

[0016] In some aspects of the method, the dsRNA comprises a sequence selected from SEQ ID No: 3, 5, and 10. In some aspects, the dsRNA comprises more than one portion having 17 or more adjacent nucleotides, having a sequence having about 95% to about 100% complementarity with the target gene or a portion thereof. In some aspects, the dsRNA is blunt-ended. In some aspects, the dsRNA has a protruding end at at least one terminal. In some aspects, the dsRNA is (a) chemically synthesized, or (b) produced by expression in microorganisms, expression in plant cells, or by microbial fermentation. In some aspects, the dsRNA is chemically modified.

[0017] In some aspects of this method, contact includes applying a composition containing dsRNA to the surface of an insect or the surface of a plant infected by the insect.

[0018] In some aspects of the method, the composition comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulating agent, microbeads, carrier particles, film, matrix, or seed treatment agent. In a further aspect, contact includes providing dsRNA in the composition, said composition further comprising one or more components selected from carrier reagents, surfactants, organosilicones, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide pesticides, safeners, insect attractants, and insect growth regulators. In some aspects, contact includes providing dsRNA in the composition, said composition further comprising at least one pesticide reagent. In some aspects, the pesticide reagent is selected from potato glycoproteins, phytohemagglutinins, phytoecdysones, and insecticidal proteins. In some aspects, contact includes providing dsRNA in the composition ingested by insects. In some aspects, the ingested composition further comprises one or more components selected from carrier reagents, surfactants, organosilicones, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide pesticides, safeners, insect attractants, and insect growth regulators.

[0019] This disclosure further covers methods for causing mortality in insects, including providing an insecticidal composition or a plant disclosed herein to the insect's food, wherein, upon ingestion by the insect, the composition or plant causes mortality or developmental delay in the insect. In some respects, the insect is an adult, or in a larval or nymphal stage.

[0020] Further, methods are provided for providing plants with improved resistance to plant-infecting insects. In this respect, the method includes introducing into a plant a recombinant DNA construct expressing a nucleotide sequence encoding an RNA molecule containing a silencing element, the silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of an insect, wherein the target gene encodes an mRNA sequence selected from SEQ ID Nos: 1, 4, 6, and 8, and wherein ingestion of the RNA by the insect results in mortality or developmental retardation in the insect. In some aspects, the silencing element has a sequence having about 95% to about 100% sequence identity or complementarity with a sequence selected from SEQ ID Nos: 3, 5, and 10. In some aspects, the recombinant DNA construct further comprises a heterologous promoter operatively linked to the nucleotide sequence encoding the RNA molecule and functional in plant cells. In some aspects, the silencing element is dsRNA, miRNA, small siRNA, hpRNA, or pi-RNA. In some aspects, the plant-infecting insect is an adult, or in a larval or nymphal stage. In some aspects, the plant-infecting insect is a Coleoptera insect. In some aspects, the plant-infecting insect is an insect from the genus *Lean Tarsus*. In some aspects, the plant-infecting insect is the potato beetle (*Radiata scoparia*). In some aspects, the introduction includes transgenic expression or transient expression. In some aspects, plants produced by the disclosed methods and possessing improved resistance to insects are further provided. In further aspects, the fruit, seeds, or reproductive parts of the disclosed plant are further provided. Attached Figure Description

[0021] Figure 1 Results from RNAi screening assays involving 95 CPB essential genes (EGs) are shown. Each dsRNA was provided to detached leaves at a concentration of 120 µl, 10 ng / µL. Treatments with two dsRNAs from two genes involved mixing 10 ng / µL of each dsRNA. Appropriate concentrations of non-target dsRNA were used. As indicated above, the figure shows mortality rates on the last day of the assay. The figure shows results extracted from several assays, and assays where non-target dsRNA (negative control) treatments resulted in mortality rates greater than 20% were discarded as invalid assays.

[0022] Figures 2A-2E Results from screening assays involving LdEG12 are shown. Figure 2AThe mean percentage of larval mortality in Colorado potato beetle (CPB) larvae (L2) exposed to cut leaves treated with 120 µl of 10 ppm (10 ng / µL) LdEG12 dsRNA or non-target dsRNA (negative control) for 4 days is shown. Five L2 larvae were placed in each cup. Each treatment had 6 replicates. Mortality was scored at days 4, 5, 6, and 8, and a one-way ANOVA was run on mortality at day 8. (F) 1,10 = 75.34, p<0.001 as indicated by ***). Figure 2B The percentage mortality of larvae in CPB L2 cells exposed to 120 µl of 10 ppm (10 ng / µL) LdEG12 dsRNA or non-target dsRNA (negative control) in 150 x 25 mm culture dishes is shown. Untreated leaves were added after day 4. Five L2 larvae were placed in each dish. Each treatment had six replicates. Mortality was scored at days 4 and 7. A one-way ANOVA was run on the mean percentage mortality at day 7. (F...) 1,10 = 375.6, p<0.001 as indicated by ***). Figure 2C The percentage mortality rate of CPB larvae in 32-well bioassay trays is shown. The CPB larvae were exposed to 24 mm diameter leaf discs treated with 24 µl of 10 ppm (10 ng / µL) LdEG12 dsRNA or non-target dsRNA (negative control) for 4 days. After 4 days, untreated leaf discs were added to the trays. Mortality was scored on day 8. The experiment was repeated twice, with 4 replicates per experiment, and a one-way ANOVA was run on the percentage mortality rate on day 8. (F) 1,4 = 80.81, p<0.001 as indicated by ***). Figure 2D This figure shows the knockdown of LdEG12 gene expression following dsRNA ingestion by L2 CPB larvae. Bioassays were performed in 32-well trays, with each CPB L2 larva exposed to 24 µL of either 10 ppm (10 ng / µL) dsRNA LdEG12 or non-target dsRNA on a leaf for 4 days. After 4 days, live larvae were collected for RNA extraction and cDNA synthesis. The cDNA was used in qPCR using TaqMan master mix. LdEG12 gene expression on the figure was normalized using RPL18 and ARF1 reference gene expression. Figure 2E The study showed the mortality rate of LdEG12 compared to three other genes (all of which function in mitochondria). For this assay, the bioassay is as described above. Figure 2C-2D The procedure is performed in a 32-well tray. Lowercase letters on the strips indicate significantly different treatments, where the same letter indicates no significant difference, and different letter indications between strips / treatments produce significantly different results.

[0023] Figures 3A-3C Results from screening assays involving LdEG53 are shown. Figure 3A The larval mortality rate in CPB L2 cells exposed to 120 µl of 10 ppm (10 ng / µL) LdEG53 dsRNA or non-target dsRNA (negative control) for 4 days is shown. Each treatment had 6 replicates. One-way ANOVA was run on the mean percentage of mortality at day 8 (F1,6 = 18.44, p < 0.01 indicated by ***). Figure 3B The above text shows the information regarding... Figure 2D The LdEG53 gene was knocked down after treatment with dsRNA. Figure 3C The percentage larval mortality rate of CPB larvae at 14 days is shown in whole plants treated with 52 ppm (52 ​​ng / µL) LdEG53 dsRNA or in untreated plants. There were 5 cages / biological replicates / treatments. (ANOVA at day 14: F1,3,35=454.711, p<0.001 indicated by ***).

[0024] Figures 4A-4B The results from screening assays involving LdEG81 are shown. Figure 4A The lethality of LdEG81 in bioassays performed in a bioassay tray is shown. One CPB larva was placed in each well of a 32-well bioassay tray. Leaves were treated with 24 µl of 10 ppm dsRNA LdEG81 or non-target negative control dsRNA. Each treatment had a total of 20 insects. Figure 4B The lethality of LdEG81 in whole-plant assays is shown. Whole-plant Kennebec potato plants aged 3–5 weeks were treated with 52 ppm dsRNA LdEG81 or left untreated. Ten L2 CPB larvae were placed on the plants in butterfly cages. There were 5 cages / biological replicates / treatments. (ANOVA on day 14: F) 1,28 = 69.82, p<0.001 as indicated by ***). Detailed Implementation

[0025] This disclosure covers compositions and methods for controlling insect pests. This disclosure is partly based on the surprising discovery of LdEG12, LdEG53, and LdEG81 in the plant insect pest CPB, whose expression, when downregulated using RNAi, can lead to mortality in the insect pest (e.g., CPB). The disclosed target genes have been identified as essential genes by the inventors, and recombinant RNA molecules have been optimized as potential insecticides.

[0026] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials associated with the cited publications. The publications discussed herein are provided only for their publication prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to precedence over such publications by virtue of a prior invention.

[0027] A. Definition Before disclosing and describing the compounds, compositions, articles, systems, devices, and / or methods herein, it should be understood that they are not limited to specific synthetic methods (unless otherwise specified) or particular reagents (unless otherwise specified), as such methods or reagents can certainly vary. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, exemplary methods and materials are described herein.

[0028] This disclosure describes the inventive concept with reference to specific examples. However, it is intended to cover all modifications, equivalents, and alternatives to the inventive concept consistent with this disclosure.

[0029] As used in the specification and appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise.

[0030] The phrase "consistently composed of..." limits the scope of the claim to the components listed in the composition or the steps listed in the method, as well as those components or steps that do not substantially affect one or more basic and novel characteristics of the claimed composition or the claimed method. The phrase "consisting of..." excludes any components, steps, or elements not listed in the claim.

[0031] As used herein, the terms “comprising,” “including,” “covering,” and “having” are used interchangeably in this disclosure.

[0032] As used herein, when referring to any numerical value, the term 'about' means a value falling within ± 10% of the stated value.

[0033] A range may be expressed herein as 'about' a particular value and / or 'about' another particular value. When expressing such a range, a further aspect includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the prefix "about", it should be understood that the particular value constitutes a further aspect. It should be further understood that the endpoints of the various ranges are meaningful not only relative to another endpoint but also independent of that other endpoint. It should also be understood that numerous values ​​are disclosed herein, and each value is also disclosed herein as 'about' that particular value, in addition to the value itself. For example, if the value '10' is disclosed, then 'about 10' is also disclosed. It should also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0034] The parts by weight of a particular element or component in the composition referred to in this specification and the concluding claims indicate the weight relationship between that element or component and any other element or component in the composition or article thereof. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such a ratio regardless of whether the compound contains other components.

[0035] As used herein, the terms “optional” or “optionally” mean that the event or situation subsequently described may or may not occur, and the description includes both cases in which said event or situation occurs and cases in which it does not occur. In one aspect, the disclosed method may optionally include one or more additional steps, such as repeating the application step or changing the application step.

[0036] As used herein, the terms “or” and “and / or” should be interpreted as inclusive or referring to any one or any combination thereof. Therefore, “A, B, or C” or “A, B, and / or C” means any of the following: “A,” “B,” or “C”; “A and B”; “A and C”; “B and C”; “A, B, and C”. Exceptions to this definition occur only when the combination of elements, functions, steps, or actions is inherently mutually exclusive in a certain way.

[0037] This disclosure also considers that, in some respects, any feature or combination of features set forth herein may be excluded or omitted. For example, if the specification states that a complex comprises components A, B, and C, it specifically anticipates that any one of A, B, or C, or a combination thereof, may be omitted and declared waived, either individually or in any combination.

[0038] As used herein, the terms “nucleic acid” and “nucleotide” refer to isolated, purified, natural, recombinant, or synthetic deoxyribonucleotides or ribonucleotides and their polymers, consisting of monomers (nucleotides) containing sugars, phosphate esters, and bases that are purines or pyrimidines, in single-stranded or double-stranded form. Unless explicitly limited, the term covers nucleic acids containing known analogs of natural nucleotides, having similar binding properties to a reference nucleic acid, and being metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated reference sequence.

[0039] As used herein, the term "polynucleotide" refers to a molecule composed of two or more, preferably more than three, and usually more than ten deoxyribonucleotides or ribonucleotides. There is no precise upper limit to the size of oligonucleotides. However, in general, oligonucleotides are shorter than about 250 nucleotides, preferably shorter than about 200 nucleotides, and more preferably shorter than about 100 nucleotides. The exact size depends on a number of factors, which in turn depend on the final function or use of the oligonucleotide. Oligonucleotides can be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.

[0040] As used herein, the terms “recombinant RNA” and “recombinant nucleotide” refer to polynucleotide molecules that contain genetically modified components through manipulation via mutagenesis, restriction enzymes, etc.

[0041] In the context of two or more nucleic acid or polypeptide sequences, the term "identity" or "percentage identity" refers to two or more biological sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., having at least about 60%, preferably 65%, 70%, 75%, preferably 80%, 85%, 90%, or 95% identity) when compared and aligned with respect to maximum correspondence within a comparison window or specified region, as measured using one of the sequence comparison algorithms described below or by manual alignment and visual inspection. Such sequences having at least about 60% identity are then described as "substantially identical." This definition also refers to the complement of the test sequence. Preferably, the identity is present in a region of at least about 25 amino acids or nucleotides in length, or more preferably in a region of 50-100 amino acids or nucleotides in length. Those skilled in the art will recognize that a polynucleotide sequence need not be identical to the sequence of the gene from which it is derived, but can only be "substantially identical." For sequence comparisons, typically one sequence serves as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and a reference sequence are input into the computer. If necessary, the coordinates of the subsequences and the sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the program parameters. Non-limiting examples of algorithms suitable for determining percentage sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

[0042] As used herein, “complementary” means that the nucleotide sequence is completely complementary to the target nucleotide sequence within more than two nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more adjacent nucleotides. Further, as used herein, the term “complementary” refers to both DNA-DNA complementarity and DNA-RNA complementarity. Similarly, the term “RNA equivalent” essentially means that in a DNA sequence, the base “T” can be replaced by the corresponding base “U” normally found in ribonucleic acid.

[0043] As used herein, "effective amount" and "effective quantity" can refer to an amount sufficient to achieve the desired result, such as the control of plant pests. In some aspects, the compositions and / or methods described herein can be used curatively, preventively, or systematically to control insect pests, or to prevent the growth, development, infection, or infestation of insect pests. Therefore, the present invention can allow previously susceptible organisms to develop resistance to infestation by insect organisms.

[0044] As used herein, insect control encompasses the control of insect offspring (e.g., egg development, for example, in the case of insect pests), the inhibition of insect vitality, growth, development, or reproduction, or the reduction of insect pathogenicity or infectivity. In some respects, insect control can inhibit biological activity in insects, resulting in one or more of the following properties: reduced insect feeding, reduced insect vitality, insect death, inhibition of insect differentiation and development, and the absence or reduction of the following abilities: sexual reproduction, muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, spermatogenesis, pheromone synthesis, pheromone sensing, antennal formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, nerve transmission, cell division, energy metabolism, respiration, apoptosis, and any component of the cytoskeleton structure of eukaryotic cells, such as actin and tubulin.

[0045] As used herein, “pest” or “insect pest” refers to an organism that has characteristics that are considered harmful or undesirable. In some respects, pests cause significant plant loss, plant damage, any destruction or loss of value, usefulness, or capability resulting from behavior or events associated with pests, such as insects. Types of plant damage include, but are not limited to, the following: Feeding damage occurs due to direct feeding on the above-ground and / or underground parts of the plant; holes or gaps in leaves and other plant parts; leaf hollowing (removal of interveinal tissue); leaf drop; cutting the plant at the soil surface; or root consumption can all occur due to pests with chewing mouthparts. Chewing pests can also burrow into plant tissue or dig tunnels within it. Stem-boring insects can kill individual stems or the entire plant or deform it. Leaf-mining insects feed between the upper and lower surfaces of leaves, creating unique tunnel-like patterns visible as translucent lines or patches on the leaves. Pests with piercing-sucking mouthparts can extract sap from plant tissue, which can lead to spots or punctate markings on leaves, leaf curling, and delayed or deformed fruit development. Insects such as thrips have rasping-sucking mouthparts that scrape the surface of leaves or flower parts, damaging plant cells. Oviposition damage occurs due to eggs being laid inside plant tissue. Insects can cause the death of stems or branches or the death of terminal shoots by laying a large number of eggs inside the stem. Flagging is the result of the death of terminal shoots at the ends of stems or branches. Laying eggs in fruit can lead to deformed or aborted fruit, sometimes called cat-facing fruit. Some insects form galls on their host plants, causing abnormal plant growth. Depending on the insect species, gall formation can be stimulated by feeding or by laying eggs into plant tissue. Pests can also cause damage by spreading plant pathogens such as viruses, fungi, bacteria, ursilli, protozoa, and nematodes. Spreading can be accidental or incidental (plant pathogens enter plant tissue through feeding or oviposition wounds), carried or passive (pests carry plant pathogens from one plant to another), or active (plant pathogens are carried in the insect's body, and the plant is inoculated by the pathogen when the insect feeds on the plant).

[0046] As used herein, the term "insect" refers to any insect belonging to the Kingdom Animals, more specifically the phylum Arthropoda, and the class Insecta or class Arachnida. As used herein, "insect" also encompasses insects at all developmental stages, including egg, larva or nymph, pupa, and adult stages. The compositions and methods disclosed herein are applicable to all such insects that are susceptible to gene silencing via RNA interference and are capable of internalizing double-stranded RNA from their immediate environment. The compositions and methods are also applicable to insects at any stage of their development, including intermoltary stages referred to as "instars." In some aspects, the disclosed compositions and methods can target insect eggs or live larvae. All stages of the developmental cycle (including metamorphosis in winged insects) can be targeted according to this disclosure. Thus, individual stages, such as larvae, pupae, nymphs, and other developmental stages, can be targeted.

[0047] In some respects, the insects belong to any of the following orders: Acari, Araneae, Anoplura, Coleoptera, Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera, Ephemeroptera, Grylloblatodea, Hemiptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera. Order Lepidoptera, Mallophaga, Mecoptera, Neuroptera, Odonata, Orthoptera, Phasmida, Plecoptera, Protura , Psocoptera, Siphonaptera, Siphunculata, Thysanura, Strepsiptera, Thysanoptera, Trichoptera and Zoraptera.

[0048] In some respects, insects are plant pests, and non-limiting examples include species of the genus *Nilaparvatas* (e.g., *N. lugens*, the brown planthopper); species of the genus *Laodelphax* (e.g., *L. striatellus*, the small brown planthopper); species of the genus *Nephotettix* (e.g., *N. virescens* or *N. cincticeps*, the large green leafhopper, or *N. nigropictus*, the rice leafhopper); species of the genus *Sogatella* (e.g., *S. furcifera*, the white-backed planthopper); and species of the genus *Blissus*. (e.g., *B. leucopterus leucopterus* (wheat stink bug)); *Scotinophora* spp. (e.g., *S. vermidulate* (rice black bug)); *Acrosternum* spp. (e.g., *A. hilare* (green stink bug)); *Parnara* spp. (e.g., *P. guttata* (rice leafhopper)); *Chilo* spp. (e.g., *C. suppressalis* (rice stem borer), *C. auricilius* (golden-edged stem borer), or *C. polychrysus* (dark-headed stem borer) Species of the genus *Chilotraea* (e.g., *C. polychrysa*, rice stalkborer); species of the genus *Sesamia* (e.g., *S. inferens*, pink rice borer); species of the genus *Tryporyza* (e.g., *T. innotata*, white rice borer or *T. incertulas*, yellow rice borer); species of the genus *Cnaphalocrocis* (e.g., *C. medinalis*, rice leafroller); species of the genus *Agromyza* (e.g., Japanese rice leafroller).Species of the genera *Diatraea* (e.g., *D. saccharalis*, *D. grandiosella*, *Narnagus*, *Xanthodes* (e.g., *X. transversa*, *Spodoptera*, *Spodoptera littoralis*, *S. frugiperda*, *S. exigua*, *S. littoralis*, or *S. western yellow-striped armyworm*.) * *Pseudaletia* (Western Yellow-striped Armyworm); *Mythimna* spp. (e.g., armyworm *Mythmna(Pseudaletia) seperata*); *Helicoverpa* spp. (e.g., corn ear borer *H. zea*); *Colaspis* spp. (e.g., grape leaf beetle *C. brunnea*); *Lissorhoptrus* spp. (e.g., rice weevil *L. oryzophilus*); *Echinocnemus* spp. (e.g., rice weevil *E. squamos*); *Diclodispa* spp. (e.g., rice beetle *D. armigera*); *Oulema* spp. spp.) (e.g., rice leaf beetle (O. oryzae); rice weevil species (Sitophilus spp.) (e.g., rice weevil (S. oryzae)); rice gall midge species (P. oryzae); rice leafminer species (Hydrellia spp.) (e.g., wheat leafminer (H. griseola) or rice stemminer (H. sasakii)); rice stemminer species (Chlorops spp.) (e.g., rice stemminer (C.)).* *D. oryzae* (rice straw fly); *Diabrotica* spp. (e.g., *D. virgifera virgifera*, *D. barberi*, *D. undecimpunctata howardi*, *D. virgifera zeae*, *D. balteata*); *Ostrinia* spp. (e.g., *O. nubilalis*); *Agrotis* spp. (e.g., *A. ipsilon*). (Black root borer); Species of the genus *Elasmopalpus* (e.g., *E. lignosellus*, the corn seedling borer); Species of the genus *Melanotus* (wireworms); Species of the genus *Cyclocephalaspp.* (e.g., *C. borealis*, the northern masked beetle, or *C. immaculata*, the southern masked beetle); Species of the genus *Popillia* (e.g., *P. japonica*, the Japanese masked beetle); Species of the genus *Chaetocnemas* (e.g., *C. pulicaria*, the corn flea beetle); Species of the genus *Sphenophorus* (e.g., *S. maidis*, the corn weevil). (Corn weevil)); *Rhopalosiphum* spp. (e.g., *R. maidis*, corn leaf aphid); *Anuraphis* spp. (e.g., *A. maidiradicis*, corn root aphid); *Melanoplus* spp. (e.g., *M. femurrubrum*, *M. differentialis*, or *M. sanguinipes*, migratory grasshopper); *Hylemya* spp. (e.g., corn seed fly).platura (seed fly); thrips species (Anaphothrips spp.) (e.g., yellow thrips (A. obscrurus)); fire ant species (Solenopsis spp.) (e.g., thieving ants (S. milesta)); or genus species (e.g., two-spotted spider mite (T. urticae), red spider mite (T. cinnabarinus)); bollworm species (e.g., cotton bollworm (H. zea) or cotton bollworm (H. armigera)); Pectinophora species (e.g., red bollworm (P. gossypiella)); borer moth species (Earias spp.) (e.g., green-striped borer moth (E. vitella)); Heliothis species spp.) (e.g., *H. virescens* (tobacco budworm)); *Anthonomus* spp. (e.g., *A. grandis* (cotton boll weevil)); *Pseudatomoscelis* spp. (e.g., *P. seriatus* (cotton jumping mirid bug)); *Trialeurodes* spp. (e.g., *T. abutiloneus* (winged whitefly), *T. vaporariorum* (greenhouse whitefly)); *Bemisia* spp. (e.g., *B. argentifolii* (silverleaf whitefly)); *Aphis* spp. (e.g., *A. gossypii* (cotton aphid)). aphid); *Lygus* spp. (e.g., *L. lineolaris* or *L. hesperus*); *Euschistus* spp. (e.g., *E. conspersus*); *Chlorochroa* spp. (e.g., *C. sayi*); *Nezara* spp. (e.g., *N. viridula*); *Thrips* spp. (e.g., *T. tungus*).*Thaci* (Onion Thrips); *Frankliniella* spp. (e.g., *F. fusca* (Tobacco Thrips) or *F. occidentalis* (Western Thrips)); *Leptothrix* spp. (e.g., *Lactuca juncta* (False Potato Beetle) or *L. texana* (Texas False Potato Beetle)); *Lema* spp. (e.g., *L. trilineata* (Three-lined Potato Beetle)); *Epitrix* spp. (e.g., *E. cucumeris* (Potato Flea Beetle)). Flea beetles (e.g., *E. hirtipennis* or *E. tuberis*); species of the genus *Epicauta* (e.g., *E. vittata*); species of the genus *Phaedon* (e.g., *P. cochleariae*); species of the genus *Epilachna* (e.g., *E. varivetis*); species of the genus *Acheta* (e.g., *A. domesticus*); and species of the genus *Empoasca* (e.g., *E. fabae*). (Potato leafhopper); Species of the genus *Myzus* (e.g., peach aphid *M. persicae* (greenpeachaphid)); Species of the genus *Paratrioza* (e.g., potato psyllid *P. cockerelli* (psyllid)); Species of the genus *Conoderus* (e.g., southern potato click beetle *C. falli* (southern potato wireworm) or tobacco wireworm *C. vespertinus* (tobacco wireworm)); Species of the genus *Phthorimaea* (e.g., potato borer moth *P.*).Species of the genera *Operculella* (potato tuber moth); *Macrosiphum* spp. (e.g., *M. euphorbiae*, potato aphid); *Thyanta* spp. (e.g., *T. pallidovirens*, red-shoulderedstinkbug); *Phthorimaea* spp. (e.g., *P. operculella*, potato tuber moth); *Helicoverpa* spp. (e.g., *egH zea*, tomato cutworm); *Keiferia* spp. (e.g., *K. lycopersicella*, tomato pinworm); *Limonius* spp. (wireworm); *Manduca* spp. (e.g., *M. sexta* (tobacco hornworm) or *M. quinquemaculata* (tomato hornworm)); species of the genus *Liriomyza* (e.g., *L. sativae*, *L. trifolli*, or *L. huidobrensis* (leafminer)); species of the genus *Drosophila* (e.g., *D. melanogaster*, *D. yakuba*, *D. pseudoobscura*, or *D. simulans*); species of the genus *Carabus* (e.g., *C. granulatus*); species of the genus *Chironomus* (e.g., *C. tentanus*); species of the genus *Ctenocephalides*. spp.) (e.g., Cat flea (C. felis)); Species of the non-ear weevil genus (Diaprepes spp.) (e.g., root weevil (D. abbreviatus)); Species of the tooth beetle genus (Ips spp.) (e.g., pine tooth beetle (I. pini)); Species of the flour beetle genus (Tribolium spp.) (e.g., red floor beetle (T. castaneum)); Species of the tsetse fly genus (Glossina spp.) (e.g., tsetse fly (G.)).Species of the genus *Anopheles* (e.g., *Anopheles gambiae*, the malaria mosquito); species of the genus *Bollworm* (e.g., the African bollworm); species of the genus *Acyrthosiphon* (e.g., the pea aphid); species of the genus *Apis* (e.g., the western honeybee); species of the genus *Homalodisca* (e.g., the glass leafhopper); species of the genus *Aedes* (e.g., the yellow fever mosquito); species of the genus *Bombyx* (e.g., the silkworm); and species of the genus *Locusta*. (e.g., migratory locust (L. migratoria)); Boophilus spp. (e.g., microplus boa micropic)); Acanthoscurria spp. (e.g., red-haired chocolate-colored bird eater (A. gomesiana)); Diploptera spp. (e.g., Pacific beetle cockroach (D. punctata)); Heliconius spp. (e.g., red passionflower butterfly (H. erato) or red-banded butterfly (H. melopomene)); Curculio spp. (e.g., acorn weevil (C. glandium)); Plutella spp. Species of the genus *Amblyomma* (e.g., *P. xylostella*, diamondback moth); species of the genus *Amblyomma* (e.g., *A. variegatum*, cattle tick); species of the genus *Anteraea* (e.g., *A. yamamai*, silkworm moth); and species of the genus *Armigeres* (e.g., *A. subalbatus*).

[0049] In other respects, the compositions and methods disclosed herein are applicable to species of the genus *Chrysomelidae*. *Chrysomelidae* belongs to the family Chrysomelidae or leaf beetles. There are more than 30 species in the genus *Chrysomelidae*. Leaf beetles such as flea beetles and corn rootworms, and weevils such as the alfalfa weevils, are particularly important pests. Flea beetles include a large number of small leaf-eating beetles that feed on the leaves of various grasses, cereals, and herbaceous plants. Flea beetles include numerous genera (e.g., *Attica*, *Apphthona*, *Argopistes*, *Disonycha*, *Longitarsus*, *Prodagricomela*, *Systena*, and *Phyllotreta*). The flea beetle, *Phyllotreta cruciferae*, also known as the rapeseed flea beetle, is a particularly important pest. Corn rootworms include species found in the genus *Phyllotreta* (e.g., *D. undecimpunctata*, *D. longicornis*, *D. virgifera*, and *D. virgifera*). Corn rootworms cause widespread damage to corn and cucurbits. The western spotted cucumber beetle, *D. virgifera*, is a cucurbit pest in the western United States. The clover weevil (also known as the clover nose weevil) belongs to the genus *Hypera* (including *H. postica*, *H. brunneipennis*, *H. nigrirostris*, *H. punctata*, and *H. meles*) and is considered a significant pest of leguminous plants. The Egyptian clover weevil (*H. brunneipennis*) is a major pest of alfalfa in the western United States.

[0050] In some respects, the disclosed plant insect pests are selected from the following: species of the genus *Lean Tarsor* (e.g., *Potato Beetle* (Colorado Potato Beetle), *False Potato Beetle* (False Potato Beetle), or *Texas Potato Beetle* (Texas False Potato Beetle)); species of the genus *Brown Planthopper* (e.g., *Brown Planthopper* ( N. lugens (brown planthopper); species of the genus *Gnaphalium* (e.g., *Gnaphalium*). L. striatellus(small brown planthopper); species of the genus *Hemibarbus* (e.g., *Hemibarbus formosanus* or *Hemibarbus davidii*, or *Hemibarbus spp.*); species of the genus *Hemibarbus* (e.g., *Hemibarbus spp.*). S. furcifera (White-backed planthopper); species of the genus *Potamogeton* (e.g., rice stem borer, Taiwan rice stem borer) or C. polychrysus (Black-headed stem borer)); Species of the genus *Steel borer* (e.g., *Rice stem borer* (pink rice borer)); Species of the genus *Rhizophora* (e.g., *Rhizophora oryzae* (white rice borer) or *Rhizophora oryzae* (yellow rice borer)); Species of the genus *Radiata* (e.g., *Radiata spp.* (western corn rootworm), *Radiata spp.* (northern corn rootworm), *Radiata spp.* (southern corn rootworm), *Radiata spp.* (southern corn rootworm), *Radiata spp.* (Mexican corn rootworm); Species of the genus *Ceratophora* (European corn borer) O. nubilalis (European cornborer); Thrips species (e.g., yellow thrips (grass thrips)); Pectinophora Species of the genus *Bollworm* (e.g., *Bollworm*); species of the genus *Burmese moth* (e.g., *Burmese budworm*); species of the genus *Whitefly* (e.g., *Whitefly spp.*, *Greenhouse whitefly*). T. vaporariorum (Greenhouse whitefly); species of the genus *Aphis* (e.g., silverleaf whitefly). B. argentifolii (Silverleaf whitefly); Aphid species (e.g., cotton aphid) A. gossypii (cotton aphid); species of the genus *Miscanthus* (e.g., American pasture mirid bug or bean pod mirid bug); species of the genus *Miscanthus* (e.g., spotted stink bug); Chlorochroa Species of the genus *S. serratus* (e.g., *S. serratus*); species of the genus *Green bug* (e.g., *Rhizophora serratus*); species of the genus *Thrips* (e.g., *Thrips serratus*); species of the genus *Thrips* (e.g., *Thrips serratus* or *Thrips serratus*); species of the genus *Aphid* (e.g., *Aphids serratus*). M. persicae (Peach aphid); Species of the genus *Leptochloa* (e.g., potato aphid); Species of the genus *Spodoptera* (e.g., wheat bug); Species of the genus *Pseudo-green bug* (e.g., green bug). Chilotraea Species of the genus (e.g., rice straw borer) C. polychrysa (rice stalk borer); species of the genus *Rice weevil* (e.g., *Rice water weevil*).L. oryzophilus (rice water weevil); species of the genus *Aphis* (e.g., corn aphid). R. maidis (corn leafaphid); and species of the genus *Rhizophora* (e.g., corn root aphid).

[0051] In some respects, this disclosure covers compositions and methods for controlling species of the genus *Lean Tarsoria*, including the Colorado potato beetle (*Potato beetle*). Leptinotarsa ​​decemlineata (Say)) and false potato beetle (false potato beetle ( Leptinotarsa ​​juncta (Say)). In some respects, the disclosed compositions and methods are used to control the Colorado potato beetle (CPB).

[0052] As used herein, "target host" or "host plant" refers to a plant infested by insect pests. CPB includes the following pests: native potato (Potato), other cultivated and wild tuberous and non-tuberous potato species (e.g., S. demissum, S. phureja ao), and other Solanaceous plant species, with non-limiting examples including: crop species tomato (several Lycopersicon species), eggplant (Solanum melongena), pepper (several Capsicum species), tobacco (several Nicotiana species, including ornamental plants), and ground cherry (Physalis species); weed / herbaceous plant species, such as the American black nightshade (S. phureja ao). The following species are listed: carolinense, black nightshade, belladonna (species of the genus Atropa), datura (species of the genus datura), henbane (species of the genus Hyoscyamus), and buffalo thorn (species of yellow thorn nightshade (S. rostratum)).

[0053] As used herein, “plant” encompasses any plant material intended to control insect pests (e.g., treatments to prevent or reduce insect growth and / or infestation). This includes, in particular, whole plants, seedlings, propagation or reproductive materials such as seeds, cuttings, grafts, explants, etc., as well as plant cells and tissue cultures. In some respects, the plant material should express or have the ability to express: an RNA molecule comprising at least one nucleotide sequence that is at least a partial RNA complement or an RNA equivalent representing the sense strand of at least one target gene of the pest organism, such that the RNA molecule is absorbed by the pest after a plant-pest interaction, and the RNA molecule is capable of inhibiting or downregulating the expression of the target gene through RNA interference.

[0054] As used herein, the phrases “downregulation of gene expression,” “inhibition of gene expression,” and “knockdown” refer to a measurable or observable reduction in gene expression, or a detectable complete elimination of gene expression, at the level of protein products and / or mRNA products from the target gene. Preferably, the downregulation does not substantially directly inhibit the expression of other genes in the insect. The downregulation effect of the recombinant RNA molecule on gene expression can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% when compared to insects that have not been exposed to the recombinant RNA molecule, or to insects that have been exposed to the recombinant RNA molecule (which does not have any complementary target RNA present in the insect). For example, the non-insect control recombinant RNA can be RNA derived from the genome of white spot syndrome virus (WSSV). Depending on the nature of the target gene, downregulation or inhibition of gene expression in insect cells can be confirmed by: phenotypic analysis of cells or the whole insect, or by using molecular techniques to measure mRNA or protein expression, such as RNA solution hybridization, PCR, quantitative reverse transcription polymerase chain reaction (RT-qPCR), nuclease protection, RNA blotting, reverse transcription, gene expression monitoring with microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, or fluorescence activated cell analysis (FACS).

[0055] In some respects, downregulation of the target gene leads to insect mortality, or cessation or retardation of insect reproduction or growth. In some respects, insects exposed to the disclosed composition may experience at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher insect mortality compared to insects not exposed to the disclosed composition or exposed to non-insect control recombinant RNA.

[0056] As used interchangeably herein, the terms “RNA interference” and “RNAi” refer to the degradation of mRNA or inhibition of protein synthesis via an endogenous pathway involving the DICER protein complex. DICER cleaves long double-stranded RNA (dsRNA) molecules into short fragments of approximately 21 nucleotides, called small interfering RNAs (siRNAs). siRNAs unfold into two single-stranded RNAs: a guest strand and a guide strand. The guest strand is degraded, while the guide strand is incorporated into the RNA-induced silencing complex (RISC). Minimal ribonucleic acids (miRNAs) are typically 22 nucleotides long and are therefore very similar in size to siRNAs; however, miRNAs are cleaved from precursor molecules containing polynucleotide “loops” connecting the guest and guide strands, and these can be similarly incorporated into the RISC. Posttranscriptional gene silencing occurs when the guide strand specifically binds to a complementary mRNA molecule and induces cleavage via the catalytic component of the RISC, Argonaute. One possibility for the generation of siRNAs in cells is the expression of precursor RNAs called hairpin RNAs (hp-RNAs). Transcription of hpRNA is typically driven by promoters of RNA polymerase III, such as the U6 or H1 promoter, or by T7 RNA polymerase. RNA-mediated silencing utilizes inverted repeats of nucleic acids or portions thereof (in this case, substantially adjacent nucleotide segments of any nucleic acid derived from the target gene, or capable of encoding a direct homolog, paralog, or homolog of a protein of interest), preferably forming hairpin structures. The inverted repeat is cloned into an expression vector containing a control sequence. Non-coding DNA nucleic acid sequences (spacers, such as matrix attachment region fragments (MAR), introns, multi-linkers, etc.) are positioned between the two inverted nucleic acids forming the inverted repeat. Following transcription of the inverted repeat, a chimeric RNA with a (partial or complete) self-complementary structure is formed. This double-stranded RNA structure is called hairpin RNA (hpRNA). hpRNA is processed into siRNA by insects, which is incorporated into the RNA-induced silencing complex (RISC). The RISC then cleaves the target mRNA transcript, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide.

[0057] "Recombinant RNA molecules" can be RNA molecules with silencing elements and encompass interfering RNA molecules that can downregulate the expression of target insect pest genes. In some respects, recombinant RNA molecules are specific to all or any sufficiently large portion of the target insect gene and can lead to the degradation of the mRNA encoded therefrom. In some respects, recombinant RNA molecules can be dsRNA, miRNA, siRNA, or hpRNA or short hairpin RNA (shRNA). In some respects, dsRNA, siRNA, shRNA, miRNA, and / or hpRNA are complementary to the coding and / or non-coding sequences of the target gene in the insect and lead to sequence-specific inhibition of the expression of the target gene product to achieve at least partial control of Coleoptera pests. In some respects, dsRNA, siRNA, shRNA, miRNA, and / or hpRNA can be produced in vitro or in vivo by genetically modified organisms such as plants or bacteria.

[0058] As used herein, “silent RNA” or “silent RNA molecule,” also referred to herein as “repressive RNA” or “repressive RNA molecule,” indicates any RNA molecule that, upon introduction into a host cell (e.g., insect cell), reduces the expression of a target gene, particularly through transcriptional and / or post-transcriptional silencing. Such silent RNA can be, for example, “antisense RNA,” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having at least 95% sequence identity with the complementary sequence of the target nucleic acid (preferably the coding sequence of the target gene). However, antisense RNA can also target regulatory sequences of the target gene, including promoter sequences as well as transcription termination signals and polyadenylation signals. Silence RNA further includes so-called “sense RNA,” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having at least 95% sequence identity with the target nucleic acid sequence. Sense RNA can also target regulatory sequences of the target gene, including promoter sequences as well as transcription termination signals and polyadenylation signals.

[0059] As used herein, “piRNA” or “Piwi-interacting RNA” is a class of small RNAs approximately 24-31 nucleotides in length. Although both siRNA and miRNA require double-stranded RNA (unstructured long dsRNA or hairpin form, etc.), piRNA is generated from a single-stranded precursor sequence that can be antisense for the target mRNA (e.g., target mRNA of insect pests).

[0060] As used herein, “contact” refers to bringing target insect pests into contact with recombinant RNA molecules, compositions containing recombinant RNA molecules, or cells expressing recombinant RNA molecules to allow the recombinant RNA molecules to be ingested by the insect pests. In some respects, cells expressing recombinant RNA molecules may be associated with food components of insect pests to increase the uptake of recombinant RNA molecules by the insect pests. Host cells expressing recombinant RNA molecules may also be incorporated into the medium in which the insect pests grow, or into or on materials or substrates such as plant parts infected by insect pests, or impregnated into substrates or materials susceptible to insect pest infestation.

[0061] As used herein, "target gene" refers to a gene of interest in an insect that can be downregulated. In some aspects, the target gene described herein is LdEG12, LdEG53, LdEG81, or any combination thereof. In some aspects, the recombinant RNA molecule contains a target region or target nucleotide in the target insect gene and may be any suitable region or nucleotide sequence within the target LdEG12, LdEG53, or LdEG81. The target region may contain at least 17, at least 18, or at least 19 consecutive nucleotides of the target gene, more preferably at least 20 or at least 21 nucleotides of the target gene, and even more preferably at least 22, 23, or 24 nucleotides.

[0062] As used herein, an "essential gene" is a selected gene that is essentially involved in the growth, development, vitality, infectivity, and / or reproduction of an insect. In some respects, an essential gene is a gene that is likely involved in a certain biological function within an insect pest, which is essential for life, or necessary for ensuring health and vitality, or is likely involved in cell integrity, cell maintenance, reproductive capacity, etc.

[0063] As used in this article, "Potato Beetle Essential Gene 12" or "LdEG12" refers to the gene ATP synthase, β subunit (ATPsynβ). This gene is encoded by the nuclear genome and functions in mitochondria. The final step in oxidative phosphorylation is chemiosmosis mediated by complex V (also known as the ATP synthase complex or F1F0-ATPase / synthase). Multiprotein ATP synthase complexes are also found in bacteria and chloroplasts. The holoenzyme consists of two structural domains, F1 and F0. The F1 domain contains the catalytic core and is located on the outer side of the membrane, while the hydrophobic F0 domain is an embedded structural unit within the membrane. These two complexes are linked together by a central stalk and a peripheral stalk. The total molecular weight of the F1 complex is approximately 350 kDa, and its catalytic core consists of three α subunits and three β subunits. Interestingly, the α subunits are non-catalytic, while the three β subunits perform the catalytic function of the enzyme complex. In addition to the α / β subunits, three other globular proteins, γ, δ, and ε, are also part of the F1 domain. ATP synthesis in the F1 domain is coupled to proton translocation via a rotational mechanism of the central stalk.

[0064] As used in this article, “potato beetle essential gene 53” or “LdEG53” refers to gene adaptor protein complex 1, γ subunit (AP-1γ). Adaptors are heterotetrameric protein complexes. Five AP complexes, AP1–5, have been identified in mammals, while AP-4 and AP-5 are absent in insects. Each AP complex has two large subunits (one each of γ / α / δ / ε / ζ and β1–5), one medium-sized subunit (μ1–5), and one small subunit (σ1–5). AP-1, AP-2, and AP-3 are clathrin-associated complexes, while AP-4 and AP-5 are not. The AP-1 complex is located at the trans-Golgi reticulum (TGN) and rough endoplasmic reticulum (RE) and mediates the biosynthesis of secretory granules and bidirectional transport between these two membrane systems. The primary function of the AP-2 complex is clathrin-dependent endocytosis from the plasma membrane. AP-3 is located at the endosome and regulates transport between the endosomal and lysosomal pathways. For membrane recruitment, binding to phosphatidylinositol (i.e., PI4P) is sufficient for the AP-2 complex but insufficient for the AP-1 complex. In addition to phosphatidylinositol binding, the AP-1 complex also requires binding to Arf1 to localize to the TGN and ER. The interaction between Arf1 and both its β1 and γ subunits is required for AP-1 recruitment at the membrane and determines the subcellular localization of the complex. The interaction between the β1 site and Arf1 is important for allosteric activation of AP-1. The γ subunit is responsible for interacting with PI4P.

[0065] As used herein, “Potato Beetle Essential Gene 81” or “LdEG81” refers to the small ribonucleoprotein particle protein SmD1 (SmD1). The main components of the spliceosome are four types of intranuclear small ribonucleoprotein particles (snRNPs) called U1, U2, U4 / U6, and U5. These snRNPs form the spliceosome structure and perform the splicing reaction. Before initiating the splicing reaction, snRNPs recognize two features on the precursor mRNA: a short conserved sequence at the exon-intron boundary and a branching point within the intron located approximately 18 to 40 nucleotides upstream of the 3′ end of the intron. snRNPs are associated with uridine (U)-rich, non-polyadenylated intranuclear small RNAs (snRNAs), which play a crucial role in spliceosome formation and subsequent splicing of the precursor mRNA. Of these snRNAs, U2, U5, and U6 form the catalytic RNA core of the active spliceosome, U1 snRNA recruits the spliceosome to the 5′ exon-intron boundary, and U4 guides U6 to the spliceosome by acting as a molecular chaperone. Each snRNP has two sets of proteins: one set is specific to the snRNP and thus regulates the function of that particular snRNP, while the second set of proteins is common to all snRNP complexes; these shared proteins are called Sm proteins. Sm proteins bind to snRNAs (except for U6 and U6 atac) at uridine-rich docking sites called Sm sites. Seven Sm proteins are described in eukaryotes: B / B′, D1, D2, D3, E, F, and G. Sm proteins form heptamer loops around U-rich motifs, which in turn stabilize the RNA structure and, when assembled, promote the binding of other U-specific proteins to the spliceosome RNP. Therefore, Sm proteins play a crucial role in spliceosome assembly.

[0066] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0067] B. Recombinant RNA molecules This disclosure covers recombinant RNA molecules that downregulate the expression of target genes in insects that infect plants. In some aspects, the recombinant RNA molecule comprises a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence that is at least about 95% to about 100% complementary to a portion of a target gene in an insect that infects plants. In some aspects, the target gene is LdEG12, LdEG53, LdEG81, or any combination thereof. In some aspects, the target gene encodes an mRNA sequence selected from SEQ ID No: 1, 4, 6, and 8.

[0068] In some aspects, the disclosed recombinant RNA molecule comprises a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity with a portion of a target gene, wherein said target gene is LdEG12. In such aspects, the target gene encodes the mRNA sequence of SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence containing about 100% identity with the sequence of SEQ ID No: 1.

[0069] In some aspects, the disclosed recombinant RNA molecule comprises a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity with a portion of a target gene, wherein said target gene is LdEG53. In such aspects, the target gene encodes an mRNA sequence of SEQ ID No: 4, SEQ ID No: 6, or any combination thereof. In some aspects, the target gene encodes an mRNA sequence of at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence of about 100% identity with the sequence of SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence of at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence of about 100% identity with the sequence of SEQ ID No: 6.

[0070] In some aspects, the disclosed recombinant RNA molecule comprises a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity with a portion of a target gene, wherein the target gene is LdEG81. In such aspects, the target gene encodes the mRNA sequence of SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence containing about 100% identity with the sequence of SEQ ID No: 8.

[0071] In some respects, the recombinant RNA molecule contains at least one RNA strand having a sequence that is about 95% to about 100% identical or complementary to the sequence selected from SEQ ID NO: 3, 5 and 10.

[0072] In some aspects, the target gene is LdEG12, and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 3. In other aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 3. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 3.

[0073] In some aspects, the target gene is LdEG53, and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 5. In other aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 5. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 5.

[0074] In some aspects, the target gene is LdEG81, and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 10. In other aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 10. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 10.

[0075] In one respect, the recombinant RNA molecule contains a nucleotide sequence having SEQ ID NO: At least 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 6 15, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 915, 920, 925, 930, 935, 940, 94 5, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1010, 1015, 1120, 1125, 1130, 1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285, 1290, 1295, 1300,1305, 1310, 1315, 1320, 1325, 1330, 1335, 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, 1440, 1445, 1 450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495, 1500, 1505, 1510, 1515, 1520, 1525, 1530, 1535, 1540, 1545, 1550, 1555, 1560, 1565, 1570, 1575, 1580, 1585, 1590, 15 95, 1600, 1605, 1610, 1615, 1620, 1625, 1630, 1635, 1640, 1645, 1650, 1655, 1660, 1665, 1670, 1675, 1680, 1685, 1690, 1695, 1700, 1715, 1720, 1725, 1730, 1735, 1740, 1745, 175 0, 1755, 1760, 1765, 1770, 1775, 1780, 1785, 1790, 1795, 1800, 1815, 1820, 1825, 1830, 1835, 1840, 1845, 1850, 1855, 1860, 1865, 1870, 1875, 1880, 1885, 1890, 1895, 1900 or 1901.

[0076] In another aspect, the recombinant RNA molecule contains a nucleotide sequence having SEQ ID NO: At least 4 of the following: 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 3 10, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 915, 920, 925, 930, 935 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1010, 1015, 1120, 1125, 1130, 1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1271, or 1272 or more adjacent nucleotides.

[0077] In another aspect, the recombinant RNA molecule contains a nucleotide sequence having SEQ ID NO: At least 6 of the following numbers: 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 31 0, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 61 0, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 915, 920, 925, 930, 935, 94 0, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1010, 1015, 1120, 1125, 1130, 1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285, 1290, 12951300、1305、1310、1315、1320、1325、1330、1335、1340、1345、1350、1355、1360、1365、1370、1375、1380、1385、1390、1395、1400、1405、1410、1415、1420、1425、1430、1435、1440、1445、1450、1455、1460、1465、1470、1475、1480、1485、1490、1495、1500、1505、1510、1515、1520、1525、1530、1535、1540、1545、1550、1555、1560、1565、1570、1575、1580、1585、1590、1595、1600、1605、1610、1615、1620、1625、1630、1635、1640、1645、1650、1655、1660、1665、1670、1675、1680、1685、1690、1695、1700、1705、1710、1715、1720、1725、1730、1735、1740、1745、1750、1755、1760、1765、1770、1775、1780、1785、1790、1795、1800、1805、1810、1815、1820、1825、1830、1835、1840、1845、1850、1855、1860、1865、1870、1875、1880、1885、1890、1995、1900、1905、1910、1915、1920、1925、1930、1935、1940、1945、1950、1955、1960、1965、1970、1975、1980、1985、1990、1995、2000、2010、2015、2120、2125、2130、2135、2140、2145、2150、2155、2160、2165、2170、2175、2180、2185、2190、2195、2200、2205、2210、2215、2220、2225、2230、2235、2240、2245、2250、2255、2260、2265、2270、2275、2280、2285、2290、2295、2300、2305、2310、2315、2320、2325、2330、2335、2340、2345、2350、2355、2360、2365、2370、2385、2390、2395、2400、2405、2410、2415, 2420, 2425, 2430, 2435, 2440, 2445, 2450, 2455, 2460, 2465, 2470, 2475, 2480, 2485, 2490, 2495, 2500, 2505, 2510, 2515, 2520, 2525, 2530, 2535, 2540, 2545, 2550, 2555, 2560, 2565, 2570, 2575, 2580, 2585, 2590, 2595, 2600, 2605, 2610, 2615, 2620, 2625, 2630, 2635, 2640, 2645, 2650, 2655 2660, 2665, 2670, 2675, 2680, 2685, 2690, 2695, 2700, 2705, 2710, 2715, 2720, 2725, 2730, 2735, 2740, 2745, 2750, 2755, 2760, 2765, 2770, 2775, 2780, 2785, 2790, 2795, 2800, 2805, 2810, 2815, 2820, 2825, 2830, 2835, 2840, 2845, 2850, 2855, 2860, 2865, 2870, 2875, 2880, 2885, 2890, 2995, 290 0, 2905, 2910, 2915, 2920, 2925, 2930, 2935, 2940, 2945, 2950, ​​2955, 2960, 2965, 2970, 2975, 2980, 2985, 2990, 2995, 3000, 3010, 3015, 3120, 3125, 3130, 3135, 3140, 3145, 3150, 3155, 3160, 3165, 3170, 3175, 3180, 3185, 3190, 3195, 3200, 3205, 3210, 3215, 3220, 3225, 3230, 3235, 3240, 3245, 32 50, 3255, 3260, 3265, 3270, 3285, 3290, 3295, 3300, 3305, 3310, 3315, 3320, 3325, 3330, 3335, 3340, 3345, 3350, 3355, 3360, 3365, 3370, 3375, 3380, 3385, 3390, 3395, 3400, 3405, 3410, 3415, 3420, 3425, 3430, 3435, 3440, 3445, 3450, 3455, 3460, 3465, 3470, 3475, 3480, 3481, or 3482 or more adjacent nucleotides.

[0078] In another aspect, the recombinant RNA molecule comprises a nucleotide sequence having at least the following nucleotides: 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 2... 25, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 861, or 862 or more adjacent nucleotides.

[0079] In some respects, recombinant RNA molecules are double-stranded RNA (dsRNA), microRNA (miRNA), small interfering RNA (siRNA), hairpin RNA (hpRNA), or piwi-interacting RNA (pi-RNA).

[0080] In some cases, the target gene is LdEG12, and the recombinant RNA molecule is dsRNA, miRNA, siRNA, hpRNA, or pi-RNA. In other cases, the target gene is LdEG53, and the recombinant RNA molecule is dsRNA, miRNA, siRNA, hpRNA, or pi-RNA. In still other cases, the target gene is LdEG81, and the recombinant RNA molecule is dsRNA, miRNA, siRNA, hpRNA, or pi-RNA.

[0081] In one respect, recombinant RNA molecules are double-stranded RNA. dsRNA may contain an RNA strand containing, for example, 800-1700 nucleotides of SEQ ID NO: 1, 1000-1700 nucleotides of SEQ ID NO: 1, 1197-1546 nucleotides of SEQ ID NO: 1, 500-1300 nucleotides of SEQ ID NO: 4, 600-1200 nucleotides of SEQ ID NO: 4, 700-1200 nucleotides of SEQ ID NO: 4, 805-1155 nucleotides of SEQ ID NO: 4, 1000-1800 nucleotides of SEQ ID NO: 6, 1100-1700 nucleotides of SEQ ID NO: 6, 1283-1633 nucleotides of SEQ ID NO: 6, 100-600 nucleotides of SEQ ID NO: 8, 200-500 nucleotides of SEQ ID NO: 8, or 204-495 nucleotides of SEQ ID NO: 8.

[0082] In some respects, the recombinant RNA molecule is a dsRNA containing an RNA chain having a sequence selected from SEQ ID NO:3, 5 and 10.

[0083] In some aspects, the target is LdEG12, and the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0084] In some aspects, the target is LdEG53, and the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0085] In some aspects, the target is LdEG81, and the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0086] In some respects, the double-stranded RNA (dsRNA) is preferably about 17-1500 base pairs (bp), about 80-1000 bp, and most preferably about 17-27 bp or about 80-250 bp; for example, double-stranded RNA regions of about 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 27 bp, 50 bp, 80 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 900 bp, 100 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, or 1500 bp. In some aspects, the disclosed dsRNA is at least 17 bp in length. In further aspects, the disclosed dsRNA contains at least 18, 19, 20, 21 or more bp in length. In some aspects, the disclosed dsRNA contains at least 22, 23 or 24 bp in length. In some aspects, the minimum length of the dsRNA is preferably at least about 80-100 bp in order to be effectively absorbed by certain insect pests.

[0087] Although dsRNA contains a sequence that corresponds to the target region of the target gene, the dsRNA as a whole does not absolutely need to correspond to the target region sequence. For example, dsRNA can contain short non-target regions flanked by target-specific sequences, provided that such sequences do not substantially affect the dsRNA's performance in RNA repression.

[0088] In some respects, dsRNA may contain one or more substituted bases to optimize performance in RNAi. It is apparent to those skilled in the art how to sequentially change each base of the dsRNA and test the activity of the resulting dsRNA (e.g., in a suitable in vitro assay system) to optimize the performance of a given dsRNA.

[0089] In some respects, dsRNA can be further modified with DNA bases, non-natural bases, or non-natural backbone bonds or sugar-phosphate backbones, for example, to enhance stability during storage or to enhance resistance to nuclease degradation.

[0090] In some respects, double-stranded RNA can be fully double-stranded (blunt-ended) or partially double-stranded. In some respects, partially double-stranded RNA may include short single-stranded overhangs at one or both ends of the double-stranded portion, provided that the RNA can still be absorbed by the insect and guide RNAi. In some respects, double-stranded RNA may also contain internal non-complementary regions.

[0091] In some respects, this disclosure covers the simultaneous or sequential contact of two or more different dsRNAs with the same insect to achieve downregulation or repression of multiple target genes or more potent repression of a single target gene. In other respects, a dsRNA can target multiple target gene sequences. In some respects, the dsRNA region in the RNA construct can be complementary to the same or different target genes, and / or the dsRNA region can be complementary to targets from the same or different insect species.

[0092] In some aspects, the target is LdEG12, and the recombinant RNA molecule is a siRNA comprising an RNA chain having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA chain having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0093] In some aspects, the target is LdEG53, and the recombinant RNA molecule is a siRNA comprising an RNA chain having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA chain having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0094] In some aspects, the target is LdEG81, and the recombinant RNA molecule is a siRNA comprising an RNA chain having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA chain having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0095] In some respects, the siRNAs disclosed herein have a length ranging from 19 to 25 base pairs, or from 20 to 24 base pairs. In some respects, the siRNAs may have 19, 20, 21, 22, 23, 24, or 25 base pairs corresponding to the target gene to be downregulated, and in some respects, a length of 21 or 22 base pairs.

[0096] In some aspects, siRNA may include single-stranded overhangs at one or both ends of the double-stranded portion. In some aspects, siRNA may contain 3′ overhanging nucleotides, preferably two 3′ overhanging thymidine (dTdT) or uridine (UU). In some aspects, although 3′ TT or UU overhangs may also be included at the other end of the siRNA, the target sequence downstream of the sequence included in the double-stranded portion of the siRNA does not necessarily have to have AA. In such aspects, siRNAs that are RNA / DNA chimeras are also considered. These chimeras include, for example, siRNAs containing double-stranded RNA with 3′ overhangs having DNA bases (e.g., dTdT), and double-stranded RNAs that are polynucleotides in which one or more RNA bases or ribonucleotides, or even all ribonucleotides on the entire strand, are replaced by DNA bases or deoxyribonucleotides.

[0097] In some aspects, the target is LdEG12, and the recombinant RNA molecule is a miRNA comprising an RNA chain having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA chain having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0098] In some aspects, the target is LdEG53, and the recombinant RNA molecule is a miRNA comprising an RNA chain having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA chain having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0099] In some aspects, the target is LdEG81, and the recombinant RNA molecule is a miRNA comprising an RNA chain having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA chain having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0100] miRNA precursors are typically transcribed into single-stranded RNA. This single-stranded RNA includes at least one stem-loop, which can be considered equivalent to the naturally occurring precursor miRNA as it is processed into mature miRNA. A stem-loop is formed when the single strand folds back on itself and sufficient base pairing occurs to stabilize the resulting folded structure. The stem-loop comprises a stem region and a loop region entirely within the same single strand of the RNA. The stem region comprises a first segment and a second segment connected by the loop region. The first segment comprises at least 19 adjacent nucleotides for silencing messenger RNA encoding the target gene. The second segment contains at least 19 adjacent nucleotides. The first and second segments generally have similar lengths (in terms of the number of adjacent nucleotides constituting each segment), but are not necessarily the same length. The loop region is located on the single strand between the first and second segments. The first and second segments hybridize to form a partially double-stranded RNA, wherein at least one nucleotide in the first segment is unpaired; that is, within the partially double-stranded RNA, there is at least one nucleotide in the first segment that is mispaired at a corresponding position in the second segment. Mismatches result in protrusions, loops, or kinks in the stem region, which is otherwise essentially double-stranded. For example, a mismatch can be due to at least one nucleotide in the second segment that does not base-pair with the nucleotide discussed in the first segment, or at least one additional nucleotide or at least one missing nucleotide at a position in the second segment corresponding to the nucleotide discussed in the first segment.

[0101] In some aspects, the first segment of the stem region includes at least 19 adjacent nucleotides for silencing messenger RNA encoding the target gene. In some aspects, the nucleotide sequence of the at least 19 adjacent nucleotides of the first segment of the stem region is selected such that the mature miRNA processed by the stem loop is completely complementary to the target mRNA at nucleotide positions 2, 3, 4, 5, 6, and 7 (starting from the 5′ end) of the mature miRNA. In some aspects, the nucleotide sequence of the at least 19 adjacent nucleotides of the first segment of the stem region is designed such that the mature miRNA processed by the stem loop structure has a few G:U swing base pairs or no G:U swing base pairs. The loop region in the stem loop typically includes about 4 to about 40 nucleotides. In some preferred embodiments, the loop region includes consecutive nucleotides of the natural loop sequence of the invertebrate miRNA precursor. In some embodiments, the loop region is identical to the natural loop sequence of the invertebrate miRNA precursor.

[0102] In some respects, the stem-loop is processed into a mature miRNA typically 21, 22, 23, 24, 25, or 26 nucleotides in length. In other respects, the stem-loop is preferably left relatively intact (i.e., essentially uncut into smaller polynucleotides), but is processed into a mature miRNA (typically 21, 22, 23, 24, 25, or 26 nucleotides in length) in the gut or cells of the insect pest that ingests the miRNA.

[0103] In some respects, single-stranded RNAs include a single stem-loop that is processed into mature miRNAs. In other respects, single-stranded RNAs include multiple stem-loops that are processed into mature miRNAs. When multiple stem-loops are present, they can consist of multiple identical stem-loops or multiple different stem-loops. In some respects, single-stranded RNAs include multiple stem-loops corresponding to a group of invertebrate miRNAs that are naturally transcribed in a single polycistronic transcription unit.

[0104] In some aspects, the target is LdEG12, and the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0105] In some aspects, the target is LdEG53, and the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0106] In some aspects, the target is LdEG81, and the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0107] In some respects, hpRNA comprises an RNA molecule that hybridizes to form a hairpin structure, the hairpin structure comprising a single-stranded circular region and a base-paired stem. The base-paired stem region contains all or part of a sense sequence corresponding to an endogenous messenger RNA encoding a target gene, and an antisense sequence that is fully or partially complementary to the sense sequence. In some respects, the sense and antisense sequences generally have similar lengths, but may differ in length. In some respects, these sequences may be at least 10, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 70, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, or 900 nucleotides in length, or portions or fragments at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. In some respects, the length of the loop region can be at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb.

[0108] In some respects, hpRNAs can be formed when the sense and antisense regions form a double-stranded RNA region. hpRNAs can be classified as long hpRNAs with long sense and antisense regions, which can be largely complementary but do not need to be perfectly complementary (typically larger than about 200 bp, ranging from 200 to 1000 bp). hpRNAs can also be quite small, ranging from about 30 to about 42 bp, but not much longer than 94 bp.

[0109] In some respects, the length of the disclosed sense or antisense RNA can be approximately 30 nt, 50 nt, 100 nt, 200 nt, 300 nt, 500 nt, 1000 nt, 2000 nt, or even approximately 5000 nt or more. The longer the sequence, the less stringent the requirement for overall sequence identity. In some respects, the length of sense or antisense RNA is 20 to 30 nucleotides. In some respects, the length of sense or antisense RNA is 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides. In some respects, the lengths of sense and antisense RNA are the same, or differ by at most 10% or 20%, and no more.

[0110] In some aspects, the polyadenylated short hairpin RNA optionally has a sense nucleotide sequence of 20-30 linking nucleotides having at least 95% identity with a first region of 20-30 nucleotides in the target gene, and an antisense nucleotide sequence of 20-30 linking nucleotides having at least 95% identity with the complement of the first region of the target gene, the sense and antisense sequences being covalently linked by a 3-20 nucleotide spacer. The first and second regions of the target gene are preferably different, and more preferably non-overlapping. Preferably, the short hairpin RNA is expressed by a chimeric gene having an RNA polymerase III (RNA Pol III) promoter, and the miRNA molecule is transcribed as a precursor RNA (primary miRNA or precursor microRNA) by a chimeric gene having an RNA Pol II promoter, the precursor RNA being subsequently processed in the cell.

[0111] In some aspects, the target is LdEG12, and the recombinant RNA molecule is a pi-RNA comprising an RNA chain having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA chain having a complementary sequence having at least about 100% identity with SEQ ID NO: 3.

[0112] In some aspects, the target is LdEG53, and the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 5.

[0113] In some aspects, the target is LdEG81, and the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 10.

[0114] In some respects, pi-RNA may contain about 26 to 30 nucleotides complementary to the target disclosed herein. In some respects, pi-RNA may contain about 26, 27, 28, 29, or 30 nucleotides complementary to the target disclosed herein.

[0115] In some respects, recombinant RNA is provided in microbial or plant cells that express recombinant RNA, or in microbial fermentation products, or is chemically synthesized.

[0116] In some aspects, the dsRNA, siRNA, miRNA, shRNA, hpRNA, or piRNA of this disclosure can be produced by those skilled in the art using chemical or enzymatic methods via artificial or automated reactions, or in vivo in cells containing nucleic acid molecules comprising polynucleotides encoding RNA, dsRNA, siRNA, miRNA, shRNA, hpRNA, or piRNA molecules. RNA can also be produced by partial or overall organic synthesis; any modified ribonucleotides can be introduced via in vitro enzymatic or organic synthesis. RNA molecules can be synthesized by cellular RNA polymerases or bacterial phage RNA polymerases (e.g., T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase). Any expression construct known in the art for the cloning and expression of polynucleotides can be used. RNA molecules synthesized chemically or via in vitro enzymatic synthesis can be purified before introduction into cells. For example, RNA molecules can be purified from a mixture by solvent or resin extraction, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, RNA molecules synthesized chemically or via in vitro enzymatic synthesis can be used without purification or with minimal purification, for example, to avoid loss due to sample handling. The RNA molecules can be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing and / or stabilize the duplex strands.

[0117] In some aspects, this disclosure also provides DNA constructs for introduction into cells (e.g., bacterial cells, fungal cells, or plant cells), wherein the DNA construct comprises a polynucleotide that, upon expression as RNA and ingestion by an insect pest, achieves downregulation of target genes in the cells, tissues, or organs of the insect pest. In some aspects, a polynucleotide is further provided that is capable of being expressed in plant cells as dsRNA, siRNA, miRNA, shRNA, hpRNA, or pi-RNA to downregulate the expression of target genes in insect pests. In such aspects, the polynucleotide may include one or more regulatory elements operatively linked to a polynucleotide capable of being expressed as dsRNA, siRNA, miRNA, shRNA, hpRNA, or piRNA.

[0118] In some aspects, the disclosed recombinant RNA molecules or DNA constructs comprising polynucleotides for expressing recombinant RNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, or pi-RNA) are expressed by prokaryotic host cells or host organisms such as bacteria or eukaryotic host cells such as fungi. In such aspects, any bacterial or yeast cell capable of expressing recombinant RNA molecules or DNA constructs can be used. In some aspects, bacteria include Gram-negative and Gram-positive bacteria, such as, but not limited to, species of the genus *Escherichia* (…). Escherichia (e.g., Escherichia coli), species of the genus Bacillus (spp.) Bacillus (e., Bacillus thuringiensis) B. thuringiensis Rhizobium species ()) Rhizobium spp.), species of the genus Lactobacillus ( Lactobacilllus spp.), species of the genus *Lactococcus* ( Lactococcus spp.), species of the genus *Pseudomonas* ( Pseudomonas spp.), pathogenic bacillus species ( Xenorhabdus spp.) or species of the genus *Luminobacterium* ( Photorhabdus (spp.), etc. Non-limiting examples of fungi may include yeasts (Saccharomyces cerevisiae ...)). Saccharomyces cerevisiae Pichia pastoris () Pichia pastoris ) and grape juice contain Hansenula polymorpha ( Hanseniaspora uvarum ))wait.

[0119] In some respects, contact with insect pests can be provided with recombinant RNA in the form of plant, bacterial, or fungal cells containing recombinant RNA, either in the insect's food or as a fermentation product (e.g., dsRNA in hairpin form produced in bacterial or fungal cells). In such respects, the recombinant RNA is expressed in bacterial, fungal, or plant cells, and these cells are absorbed or consumed by the insect pests. In some respects, the bacterial or fungal cells that produce the recombinant RNA can be sprayed directly onto the crop.

[0120] In some respects, this document provides recombinant DNA constructs containing heterologous promoters that are functional for RNA transcript expression in bacteria or fungi. In some respects, the recombinant DNA constructs are intended for expression in bacteria selected from species such as *Escherichia coli*, *Bacillus*, *Rhizobium*, *Lactobacillus*, *Lactococcus*, *Pseudomonas*, *Pathogenic Bacillus*, or *Luminobacter*. In some respects, the recombinant DNA constructs are intended for expression in fungi selected from yeast species such as *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Hansenula polysaccharide*.

[0121] In some respects, heterologous promoters that are functional for expression of RNA transcripts in bacteria include promoters that can be induced by the use of appropriate chemicals such as IPTG, constitutive promoters, bacterial phage promoters such as T7, T3, SV40 or SP6 promoters, promoters from RNA Pol I, RNA Pol II or RNA Pol III polymerases, tac, trc and lac promoters, β-lactamase promoters, PL and PR promoters from Escherichia coli λ phage, and galactose promoters, arabinose promoters and alkaline phosphatase promoters from Escherichia coli.

[0122] Suitable promoters for expression in yeast are well known and include, for example, the bacterial phage T7 promoter, promoters from GAL1 (which are induced by the presence of galactose), ADH1, TEF1 promoters, and AOX promoters (methanol-inducible promoters). Many yeast cloning vectors have been designed and are readily available. Methods for transforming Saccharomyces cerevisiae cells from exogenous DNA to produce recombinant polypeptides are also well known. Transformed cells are selected based on phenotype determined by selectable markers, common drug resistance, or the ability to grow in the absence of specific nutrients (e.g., leucine).

[0123] In some aspects, this disclosure further covers the generation of recombinant RNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, or pi-RNA). In such aspects, recombinant RNA molecules are introduced using isolated nucleic acid or recombinant (DNA) constructs (e.g., by transformation, transfection, or injection) provided that transcription of the nucleic acid or recombinant (DNA) construct is permitted to produce recombinant RNA.

[0124] In some respects, one or more transcription termination sequences may also be incorporated into the recombinant constructs disclosed herein. Transcription termination sequences may encompass control sequences at the ends of transcription units that signal the 3′ processing and polyadenylation of the primary transcript, as well as transcription termination. Additional regulatory elements, such as transcriptional or translational enhancers, may also be incorporated into the recombinant expression constructs.

[0125] In a further aspect, the DNA construct may include an origin of replication that is essential for maintenance and / or replication in a particular cell type. For example, when the expression construct is needed to be maintained in a bacterial cell as an additional genetic element (e.g., a plasmid or kinase molecule). In some aspects, the origin of replication includes, but is not limited to, f1-ori and colE1ori.

[0126] In some aspects, the DNA construct may optionally include selectable marker genes. Selectable marker genes may include any gene that confers a phenotype on the cells in which it is expressed to facilitate the identification and / or selection of transfected or transformed cells. Non-limiting examples of selectable markers include resistance genes to ampicillin (Ampr), tetracycline (Tcr), kanamycin (Kanr), phosphatidylinosin, and chloramphenicol (CAT) genes. Other suitable marker genes provide metabolic traits, such as manA. Visual marker genes may also be used, and these include, for example, β-glucuronidase (GUS), luciferase, and green fluorescent protein (GFP).

[0127] In some aspects, viruses that specifically infect insect pests can be used to deliver recombinant RNA. In some aspects, non-limiting examples of viruses may include baculoviruses (e.g., alfalfa silver-striped moth multinucleate polyhedrosis virus), insect poxviruses (EPVs) (e.g., Amsacta moorei EPV), umbilical nuclei viruses (e.g., Aedes aegypti umbilical nuclei virus), nudiviruses, and nodaviruses (e.g., black beetle virus). Other viruses used in this invention include viruses that infect a wide range of arthropods, including white spot shrimp viruses and baculoviruses of marine shrimp, PmSNPV and PvSNPV.

[0128] In some aspects, the virus is modified using a viral transfer plasmid containing a DNA construct encoding a recombinant RNA molecule, and one or more of an enhancer, promoter, or transcription terminator. In some aspects, the DNA construct is inserted between two promoter / terminator / enhancer complexes to produce a complete expression unit capable of directing the synthesis of the recombinant RNA. The modified transfer plasmid is then used to introduce the expression unit into the viral genome.

[0129] In some respects, bacterial cells, yeast cells, or viruses can be inactivated before being applied to host plants or coming into contact with insect pests. Inactivation can be achieved by any means, such as heat treatment, treatment with phenol or formaldehyde, or mechanical treatment.

[0130] In a further aspect, this disclosure includes the step of introducing (e.g., by transformation or transfection) an isolated nucleic acid or DNA construct into a bacterial, fungal, or viral cell of this disclosure, provided that transcription of the nucleic acid or DNA construct is permitted to produce a recombinant RNA molecule. Any transformation or transfection method well-known in the art can be used to introduce an isolated nucleic acid or DNA construct into a bacterial, fungal, or viral cell.

[0131] In a further aspect, this disclosure covers the transformation of a specific host target (e.g., a plant) and recombinant DNA constructs for achieving stable transformation of the target host (e.g., a Solanaceae plant). The transformed target host can be expressed by the recombinant DNA molecule at an effective level of a recombinant RNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, or pi-RNA). In some aspects, this document further discloses plant transformation vectors comprising at least one polynucleotide operatively linked to a heterologous promoter functional in plant cells, wherein expression of said polynucleotide results in a recombinant RNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, pi-RNA).

[0132] In some aspects, polynucleotides include one or more elements selected from: (a) promoters that function in plant cells; (b) transgenic transcription units; (c) gene repression elements; and (d) transcriptional regulation / stabilization elements. A promoter can be any promoter that has promoter activity in plant cells. In some aspects, promoters can include constitutive promoters, space-specific promoters, time-specific promoters, development-specific promoters, and inducible promoters. Non-limiting examples of promoters may include spatially specific promoters, such as organelle, cell, tissue, or tissue-specific promoters (e.g., plastid-specific, root-specific, pollen-specific, or seed-specific promoters, respectively, for inhibiting the expression of the first target RNA in plastids, roots, pollen, or seeds); time-specific promoters, which may include promoters that tend to promote expression during certain developmental stages in the plant's growth cycle, or at different times of day or night, or in different seasons of the year; inducible promoters, which include promoters induced by chemicals or environmental conditions, such as, but not limited to, biotic or abiotic stresses (e.g., water shortage or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection); and expression-specific promoters, which may include promoters that are generally constitutively expressed but at different levels or "intensities" of expression, including promoters that are generally considered "strong promoters" or "weak promoters." Methods for the stable transformation of recombinant DNA molecules in plants are well known and can be used to express the disclosed polynucleotides.

[0133] In some aspects, promoter elements may include nucleic acid sequences that are not naturally occurring promoters or promoter elements or their homologs, but can regulate gene expression. Examples of such regulatory sequences include naturally occurring or artificially designed RNA sequences that include a ligand-binding region or aptamer and a regulatory region (which may be d-active). In some aspects, promoters may include riboregulators selected or designed for specific spatial or temporal specificity, for example to regulate the translation of DNA encoding a silencing element, for suppressing a target gene only in the presence (or absence) of a given concentration of an appropriate ligand. One example is a riboregulator that responds to endogenous ligands (e.g., jasmonic acid or salicylic acid) produced by plants under stress (e.g., abiotic stresses such as water, temperature, or nutrient stress, or biotic stresses such as the attachment of pests or pathogens); under stress, the level of the endogenous ligand increases to a level sufficient to cause the riboregulator to initiate DNA transcription, said DNA encoding a silencing element for suppressing a target gene.

[0134] In some aspects, the recombinant DNA construct or polynucleotide comprises DNA encoding one or more site-specific recombinase recognition sites. In one aspect, the recombinant DNA construct comprises at least a pair of loxP sites, wherein site-specific recombination of DNA between the loxP sites is mediated by Cre recombinase. The position and relative orientation of the loxP sites are selected to achieve the desired recombination; for example, when the loxP sites are in the same orientation, the DNA between the loxP sites is cleaved in a circular manner. In another aspect, the recombinant DNA construct comprises DNA encoding one loxP site; the two DNAs recombine in the presence of Cre recombinase and another DNA having a loxP site.

[0135] In a further aspect, this document provides a recombinant DNA construct comprising a heterologous promoter operatively linked to DNA encoding an RNA transcript, said RNA transcript comprising a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the RNA transcript contains 100% identity with SEQ ID NO: 3.

[0136] In some aspects, this document provides a recombinant DNA construct comprising a heterologous promoter operatively linked to DNA encoding an RNA transcript, said RNA transcript comprising a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the RNA transcript contains 100% identity with SEQ ID NO: 5.

[0137] In some aspects, this document provides a recombinant DNA construct comprising a heterologous promoter operatively linked to DNA encoding an RNA transcript, said RNA transcript comprising a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the RNA transcript contains 100% identity with SEQ ID NO: 10.

[0138] In some respects, the recombinant DNA construct contains a heterologous promoter that functions for RNA transcript expression in bacteria. In other respects, the recombinant DNA construct contains a heterologous promoter that functions in plant cells. This article further provides recombinant vectors containing recombinant DNA constructs, as well as plant chromosomes or plastids containing the disclosed recombinant DNA constructs.

[0139] This disclosure further covers transgenic plant cells having the disclosed recombinant DNA construct in their genome, as well as crop products, transgenic progeny seeds, or fertile plant parts produced from the disclosed transgenic plants.

[0140] In some respects, recombinant DNA constructs are expressed in plants via transgenic expression or transient expression. Transgenic plant cells and plants can be prepared using methods known in the art. In some respects, the recombinant DNA constructs disclosed herein can be transcribed in any plant cell or tissue or in the whole plant at any developmental stage. Methods for transient expression may include synthesizing recombinant RNA in vitro and applying it to leaves by spraying, or to roots by soaking, or abrading the surface of leaves and applying a solution containing bacterial cells expressing recombinant RNA, resulting in the transient presence of the recombinant RNA within the plant tissue.

[0141] In some respects, recombinant RNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, pi-RNA) are produced via microbial fermentation. In such respects, the disclosed recombinant DNA molecule contains a polynucleotide expressing the recombinant RNA molecule, expressed in bacteria such as *Escherichia coli*; and the resulting RNA produced in the bacteria is purified as a fermentation product.

[0142] In some aspects, when compared with gene expression in control insects unexposed to the recombinant RNA, exposure to or ingestion of the disclosed recombinant RNA results in a downregulation of LdEG12 expression in insect pests by at least 30%, 40%, 50%, 60%, preferably 70%, 80%, or even more preferably 90% or 95%. In some aspects, when compared with gene expression in control insects unexposed to the recombinant RNA, exposure to or ingestion of the disclosed recombinant RNA results in a downregulation of LdEG53 expression in insect pests by at least 30%, 40%, 50%, 60%, preferably 70%, 80%, or even more preferably 90% or 95%. In some aspects, when compared with gene expression in control insects unexposed to the recombinant RNA, exposure to or ingestion of the disclosed recombinant RNA results in a downregulation of LdEG81 expression in insect pests by at least 30%, 40%, 50%, 60%, preferably 70%, 80%, or even more preferably 90% or 95%. Gene expression can be measured using molecular techniques to measure mRNA or protein expression, such as RNA solution hybridization, PCR, RT-qPCR, nuclease protection, RNA hybridization, reverse transcription, gene expression monitoring with microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, or fluorescence activated cell analysis (FACS).

[0143] B. Insecticide Composition This disclosure further covers insecticidal compositions comprising the recombinant RNA molecule disclosed in segment B. In some aspects, the insecticidal composition comprises recombinant RNA capable of downregulating the expression of a target gene. In some aspects, the target gene is LdEG12, LdEG53, LdEG81, or any combination thereof. In some aspects, the target gene encodes an mRNA sequence selected from SEQ ID No: 1, 4, 6, and 8.

[0144] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule having a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity to a portion of a target gene of an insect infecting a plant, said target gene encoding an mRNA sequence selected from SEQ ID No: 1, 4, 6 and 8.

[0145] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule having a nucleotide sequence having at least 17 or more adjacent nucleotides, having a sequence having at least about 95% to about 100% complementarity to a portion of a target gene of an insect infecting a plant, said target gene encoding the mRNA sequence of SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence having about 100% identity with the sequence of SEQ ID No: 1.

[0146] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule having a nucleotide sequence having at least 17 or more adjacent nucleotides, having a sequence having at least about 95% to about 100% complementarity to a portion of a target gene of an insect infecting a plant, said target gene encoding the mRNA sequence of SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence having about 100% identity with the sequence of SEQ ID No: 4.

[0147] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule having a nucleotide sequence having at least 17 or more adjacent nucleotides and having a sequence having at least about 95% to about 100% complementarity to a portion of a target gene of an insect infecting a plant, said target gene encoding the mRNA sequence of SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence having about 100% identity with the sequence of SEQ ID No: 6.

[0148] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule having a nucleotide sequence having at least 17 or more adjacent nucleotides, having a sequence having at least about 95% to about 100% complementarity to a portion of a target gene of an insect infecting a plant, said target gene encoding the mRNA sequence of SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence having about 100% identity with the sequence of SEQ ID No: 8.

[0149] In some respects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising at least one RNA strand, said at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID NO: 3, 5 and 10.

[0150] In some aspects, the target gene is LdEG12, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising at least one RNA strand, said at least one RNA strand having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 3. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 3. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 3.

[0151] In some aspects, the target gene is LdEG53, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising at least one RNA strand, said at least one RNA strand having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 5. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 5. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 5.

[0152] In some aspects, the target gene is LdEG81, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising at least one RNA strand, said at least one RNA strand having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 10. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 10. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 10.

[0153] In some respects, the insecticidal composition comprises recombinant RNA molecules, including dsRNA, miRNA, siRNA, hpRNA and / or pi-RNA disclosed herein.

[0154] In some aspects, the target gene is LdEG12, and the insecticidal composition contains an insecticidally effective amount of a recombinant RNA molecule, comprising dsRNA, miRNA, siRNA, hpRNA, or pi-RNA. In some aspects, the target gene is LdEG53, and the insecticidal composition contains an insecticidally effective amount of a recombinant RNA molecule, comprising dsRNA, miRNA, siRNA, hpRNA, or pi-RNA. In some aspects, the target gene is LdEG81, and the insecticidal composition contains an insecticidally effective amount of a recombinant RNA molecule, comprising dsRNA, miRNA, siRNA, hpRNA, or pi-RNA.

[0155] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule containing dsRNA, said dsRNA comprising an RNA strand having a sequence selected from SEQ ID NO: 3, 5 and 10.

[0156] In some aspects, the target is LdEG12, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising dsRNA, said dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising dsRNA, said dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0157] In some aspects, the target is LdEG53, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising dsRNA, said dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0158] In some aspects, the target is LdEG81, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising dsRNA, said dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0159] In some aspects, the target is LdEG12, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule containing siRNA, said siRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule containing siRNA, said siRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0160] In some aspects, the target is LdEG53, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule containing siRNA, said siRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is siRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0161] In some aspects, the target is LdEG81, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule containing siRNA, said siRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is siRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0162] In some aspects, the target is LdEG12, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising miRNA, said miRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising miRNA, said miRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0163] In some aspects, the target is LdEG53, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a miRNA having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence in SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence having at least about 100% identity with the sequence in SEQ ID NO: 5.

[0164] In some aspects, the target is LdEG81, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a miRNA having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0165] In some aspects, the target is LdEG12, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising hpRNA, said hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising hpRNA, said hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0166] In some aspects, the target is LdEG53, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising hpRNA, said hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0167] In some aspects, the target is LdEG81, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising hpRNA, said hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0168] In some aspects, the target is LdEG12, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising pi-RNA, said pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising pi-RNA, said pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 3.

[0169] In some aspects, the target is LdEG53, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising pi-RNA, said pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 5.

[0170] In some aspects, the target is LdEG81, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising pi-RNA, said pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 10.

[0171] The insecticidal compositions disclosed herein may comprise at least one of the following: a recombinant RNA molecule (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA), at least one DNA molecule containing a polynucleotide for expressing the recombinant RNA molecule, and / or at least one host cell (e.g., bacteria, yeast, virus, or plant cell) expressing the disclosed recombinant RNA molecule.

[0172] In some aspects, the insecticidal composition further comprises a carrier, excipient, diluent, surfactant, organosilicon, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide pesticides, safeners, insect attractants and insect growth regulators, or any combination thereof. In further aspects, the insecticidal composition comprises a suspending agent, a flocculant, alkali, buffer, preservative, propellant, thixotropic agent, and / or antifreeze. In some aspects, the insecticidal composition may contain further components that act to stabilize recombinant RNA molecules and / or prevent the degradation of recombinant RNA molecules during extended storage of the insecticidal composition. In some aspects, the insecticidal composition may further contain components that act to preserve the viability of target host cells during extended storage.

[0173] Non-limiting examples of solid carriers or excipients may include fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, magnesium oxide powder, bleaching clay, wax, gypsum, diatomaceous earth, rubber, plastic, porcelain clay, mineral soils such as silica, silica gel, silicates, attapulgite, limestone, chalk, loess, clay, dolomite, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, plant-derived products such as cereal flour, bark flour, wood flour and nut flour, cellulose flour, attapulgite, montmorillonite, mica, vermiculite, synthetic silica and synthetic calcium silicate, or combinations thereof. Examples of liquid carriers or excipients include, but are not limited to: water; alcohols, such as ethanol, butanol, or ethylene glycol, and their ethers or esters, such as ethylene glycol methyl ether acetate; ketones, such as acetone, cyclohexanone, methyl ethyl ketone, methyl isobutyl ketone, or isophorone; alkanes, such as hexane, pentane, or heptane; aromatic hydrocarbons, such as xylene or alkylnaphthalene; mineral oils or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or dichloromethane; aromatic chlorinated hydrocarbons, such as chlorobenzene; and water-soluble or strongly polar solvents, such as dimethylformamide, dimethyl sulfoxide, or N-methylformamide. Pyrrolidone; liquefied gases; waxes, such as beeswax, lanolin, shellac wax, carnauba wax, fruit wax (e.g., wax of bayberry or sugarcane), candelilla wax, other waxes such as microcrystalline wax, ceresin, pure ceresin or lignite wax; salts such as monoethanolamine salts, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium bisulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium bicarbonate, ammonium thiosulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, sodium ammonium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamate, and mixtures thereof.

[0174] In some aspects, solid diluents may include, but are not limited to, clays such as diatomaceous earth, corn husks, tricalcium phosphate, cork powder, kaolin, bentonite, or attapulgite, as well as water-soluble polymers. In some aspects, liquid diluents include, but are not limited to, water, organic diluents, water-soluble polymers, or any combination thereof. Additionally, diluents may include, for example, ionic or nonionic wetting agents, dispersants, or emulsifiers, or mixtures thereof.

[0175] Examples of useful surfactants include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow, tallow amine, or phospholipids), and silicone surfactants. Other useful surfactants include silicone surfactants, including nonionic silicone surfactants such as Tween-20.

[0176] In some aspects, the composition may further contain components that enhance or promote the uptake of recombinant RNA molecules by insect pests. These may include, for example, chemical agents that generally promote RNA uptake into cells (e.g., lipofectamine).

[0177] In some aspects, organosilicon is an organosilicon surfactant containing a trisiloxane or modified trisiloxane structure, comprising a single organosilicon, or two or more organosilicon compounds, or organosilicon as a complex and other auxiliary agents. In some aspects, said auxiliary agents comprise APES (OP-9-15), NPE phosphate (NPEP04), APES disodium sulfosuccinate (2020), EO-PO block surfactant (EPE), castor oil polyoxyethylene ether, styrene-phenol-formaldehyde resin APEO, diphenylbiphenylaldehyde APEO, cinnamaldehyde-phenol APEO (603#), AEO (A-105), or combinations thereof.

[0178] In some respects, polynucleotide herbicidal molecules contain, for example, herbicide tolerance genes, non-limiting examples of which include genes that provide tolerance to glyphosate, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetylhydroxy acid synthase, acetyllactic acid synthase (ALS), acetyl-CoA carboxylase (ACCase), dihydropteroate synthase, phytoene desaturase (PDS), protoporphyrin IX oxidase (PPO), hydroxyphenylpyruvate dioxygenase (HPPD), para-aminobenzoic acid synthase, glutamine synthase (GS), glufosinate-resistant glutamine synthase, 1-deoxy-D-xylulose-5-phosphate (DOXP) synthase, dihydropteroate (DHP) synthase, phenylalanine aminolyase (PAL), glutathione S-transferase (GST), photosystem II D1 protein, monooxygenase, cytochrome P450, cellulase, β-tubulin, or serine hydroxymethyltransferase.

[0179] Non-limiting examples of herbicides include glyphosate, dicamba, phosphamidon, glufosinate, 2,2-dichloropropionic acid (glufosinate), acetyllactone synthase inhibitors (e.g., sulfonylureas, imidazolinones, triazolidinediones, pyrimidinyl benzoates, and phthalides), bromobenzonitrile, cyclohexanedione (haloxyfop-R-methyl), and aryloxyphenoxypropionate (flupyridine), sulfonamide herbicides, triazine herbicides, 5-methyltryptophan, aminoethylcysteine, pyridazinone herbicides (e.g., fluazinam); hydroxyphenylpyruvate dioxygenase, cyclopropylisoxazole herbicides (… Examples include isoxaflutole, protoporphyrinogen oxidase inhibitors, herbicides containing aryloxyalkylene ester moieties, phenoxy phytochemicals (e.g., 2,4-D and 2,4-D propionic acid), pyridinoxy phytochemicals (e.g., fluazinam and chlorpyrifos), aryloxyphenoxypropionate (AOPP) acetyl-CoA carboxylase (ACCase) inhibitors (e.g., flupyridine, quizalofop-P-ethyl, and haloxyfop-R-methyl), and 5-substituted phenoxyacetic acid protoporphyrinogen oxidase IX inhibitors (e.g., pyrazosulfuron and fluazinam), or any combination thereof.

[0180] In some respects, the insecticidal composition may further comprise a pesticide agent. In some respects, the pesticide agent is a potato glycoprotein, a phytohemagglutinin, a plant ecdysone, an insecticidal protein, or any combination thereof. In some respects, the pesticide agent is a chemical (organic) insecticide.

[0181] In some respects, insecticidal proteins are Bacillus thuringiensis insecticidal proteins, pathogenic bacilli insecticidal proteins, luminescent bacteria insecticidal proteins, and Bacillus laterosporus (… Bacillus laterosporous Insecticidal proteins, Bacillus spheroidae ( Bacillus sphearicus Insecticidal proteins or combinations thereof. In some respects, the insecticidal proteins are Cry1, Cry3, TIC851, CryET170, Cry22, binary insecticidal proteins CryET33 and CryET34, binary insecticidal proteins CryET80 and CryET76, binary insecticidal proteins TIC100 and TIC101, or binary insecticidal protein PS149B1.

[0182] In some respects, chemical insecticides include one or more of pyrethroids or organophosphate insecticides, including but not limited to: cypermethrin, permethrin, cypermethrin, bifenthrin, fenvalerate, deltamethrin, phosmet, methyl parathion, thiamethoxam, pyriproxyfen, flupyradifurone, acetamiprid, dinotefuran, thiamethoxam, acephate, malathion, quinalphos, chlorpyrifos, profenofos, imidacloprid, bifenthrin, chlorpyrifos, deltamethrin, diazinon, pyrethrum, cypermethrin, acetamiprid, insecticidal soaps or oils, neonicotinoids, diamides, abamectin and derivatives, spinosad and derivatives, azadirachtin, pyridaben, or any combination thereof. In some respects, chemical insecticides are insecticides that target nerves and muscles. Examples include acetylcholinesterase (AChE) inhibitors, such as carbamates (e.g., methomyl and thiamethoxam) and organophosphates (e.g., ehiorpyrifos); GABA-gated chloride channel antagonists, such as cyclodiene organochlorines (e.g., endosulfan) and phenylpyrazoles (e.g., fipronil); sodium channel modulators, such as pyrethroids and pyrethroids (e.g., cypermethrin and lambda-cyhalothrin); nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids (e.g., acetamiprid, thiamethoxam, and thiamethoxam); and nicotinic acetylcholine receptor (nAChR) agonists. Allosteric modulators, such as spinosad (e.g., spinose and ethyl spinosad), chloride channel activators, such as avermectin and milbemycin (e.g., abamectin, emamectin benzoate), nicotinic acetylcholine receptor (nAChR) blockers, such as bensuitap and fenitrothion, voltage-dependent sodium channel blockers, such as indoxacarb and cyfluthrin, and ranitidine receptor modulators, such as diamides (e.g., chlorantraniliprole and flufenoxuron). In another aspect, pesticides are pesticides that target respiration. Examples include chemicals that uncouple oxidative phosphorylation by disrupting the proton gradient, such as brofenoxuron, and mitochondrial complex I electron transport inhibitors. In some aspects, chemical pesticides are pesticides that target growth and development. Examples include juvenile hormone mimics, such as juvenile hormone analogs (e.g., phenoxycarb), type 0 inhibitors of chitin biosynthesis, such as benzoylurea (e.g., flufenoxuron, lufenuron, and flufenoxuron), and ecdysone receptor agonists, such as dihydrazides (e.g., methoxyfenozide and tebufenozide).

[0183] In some respects, the pesticide is one or more biological pesticides known to those skilled in the art. Examples of biological pesticides include, but are not limited to: azadirachtin (neem oil), toxins derived from natural pyrethroids, Bacillus thuringiensis and Beauvena bassiana, viruses (e.g., CYD-X™, CYD-X HP™, Germstar™, Madex HP™ and Spod-X™), and peptides (Spear-T™, Spear-P™ and Spear-C™).

[0184] In some respects, safeners are compounds used to reduce the phytotoxic effects of agrochemicals. Non-limiting examples of safeners in some respects include (D-4) quizalofop-p-ethyl, (D-5) bensulfuron-methyl, (D-9) chlorfluazuron, (D-11) piperazine, (D-14) chlorfluazuron, (D-15) ethyl chlorfluazuron, (D-18) furazolidone, (D-19) ethyl bis(oxazolyl)ate, (D-23) pyrazosulfuron-methyl, (D-62) N-{[4-(cyclopropylcarbamoyl)phenyl]sulfonyl}-2-methoxybenzamide (cyclopropanesulfonamide), and (D-63) N-{[4-(cyclopropylcarbamoyl)phenyl]sulfonyl}-2-methoxy-5-methylbenzamide.

[0185] In some respects, plant ecdysones include, for example, compounds comprising triterpenoids, triterpenoid saponins, phytosterols, or any combination thereof.

[0186] In some aspects, the insecticidal composition can be any suitable physical form for application to pest insects. The insecticidal composition can be in solid form, such as powder, clumps, or bait, or in liquid form, such as spray, gel, or aerosol. In some aspects, the insecticidal composition is in a form suitable for ingestion by insect pests. In some aspects, the insecticidal composition is selected from the physical forms of: solid, liquid, powder, suspension, emulsion, spray, encapsulating agent, microbeads, carrier microparticles, film, solid matrix, soil irrigation agent, insect food, insect bait, and seed treatment agent. In a further aspect, the insecticidal composition may include nanoparticles comprising a polymer matrix and recombinant RNA molecules, DNA molecules comprising polynucleotides for expressing recombinant RNA molecules, and / or at least one host cell (e.g., bacteria, yeast, or plant cells) expressing the disclosed recombinant RNA molecules. In one specific aspect, the insecticidal composition is formulated for use as a spray, feeding station, or food trap.

[0187] The nature and physical form of the excipients in an insecticidal composition can vary depending on the nature of the material or matrix to be treated. For example, the insecticidal composition can be a liquid brushed or sprayed onto or imprinted into the material or matrix to be treated, or a coating or powder applied to the material or matrix to be treated. In some aspects, the insecticidal composition is in the form of a coating on a suitable surface that adheres to and is eventually ingested by an insect pest, the insect pest coming into contact with the coating.

[0188] The preparation of powder formulations may include, for example, grinding a surfactant with a solid material such as natural clay or diatomaceous earth, such as talc, kaolin, or bentonite. Granular formulations may be prepared by spraying the active ingredient (e.g., recombinant RNA molecules) to allow it to adsorb onto inert material particles, or by applying the active ingredient to the surface of a carrier (e.g., sand, kaolin, or inert material particles) using a binder (e.g., polyvinyl alcohol, sodium polyacrylate, or mineral oil). If used with fertilizers, the recombinant RNA molecules can be prepared within the granules in the same manner as fertilizer granules.

[0189] In some respects, the host plant is treated against insect pest infestation. The insecticidal composition is then internalized or ingested by the insect, from which RNAi can be mediated, thus controlling the insect. The particles of the insecticidal composition can be of a suitable size such that they attach to the host plant to be treated or to the insect pest, such as the exoskeleton of the insect pest, and can be absorbed therefrom. The insecticidal composition can be formulated for application to the host plant, or any part of the host plant, in any suitable manner. For example, the composition can be formulated for application to the leaves, stems, roots, fruits, vegetables, grains, and / or legumes of the host plant. In some respects, the insecticidal composition is formulated for application to the leaves of a plant.

[0190] In some aspects, the insecticidal composition is in the form of a bait. The bait is designed to lure insect pests into contact with the insecticidal composition. Once in contact, the composition is then internalized by the insect pest, for example by ingestion, and mediates RNAi to thus kill the insect. The bait may further contain food matter. In some aspects, the insect food or attractant includes any type of insect food, including various sugars, proteins, carbohydrates, yeasts, fats, and / or oils. The bait may vary depending on the species being targeted. The bait may be in any suitable form, such as solid, paste, clump, or powder. In some aspects, the bait may be provided in a suitable “shell” or “trap.” Such shells and traps are commercially available, and existing traps can be modified to include the disclosed insecticidal composition.

[0191] In some respects, insecticidal attractants may also be used in the composition. The attractant may be a pheromone, such as a male or female pheromone. The attractant acts to lure insect pests to the insecticidal composition (e.g., a bait) and may target specific insect pests or attract a wide variety of insects. In some respects, the pheromone or pheromone blend comprises: methyl 2,6,10-trimethyltridecanoate, (Z)-a-bisabolene, trans- and cis-1,2-epoxides of (Z)-a-bisabolene, (E)-nerolidol, n-nonadecane, (Z)-9-tetradecene acetate, (Z,E)-9,12-tetradecadiene acetate, (Z)-11-hexadeceneal, (Z)-9-hexadeceneal, (Z)-11-hexadecene acetate, 4-methoxycinnamaldehyde, or any combination thereof.

[0192] In some aspects, the effective amount of the insecticidal composition may contain a disclosed recombinant RNA molecule (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA) in the range of about 1 ng / µl to 500 μg / ml. In some aspects, less than about 100 μg / ml of the recombinant RNA molecule / plant, particularly less than 50 μg / ml, more particularly less than about 40, 30, 20, 10, or 5 μg / ml, and most particularly less than about 1 μg / ml or about 0.5 μg / ml of the recombinant RNA molecule / plant. In some aspects, when the composition is applied to the leaves of a plant, less than about 100 μg / ml of the recombinant RNA molecule / leaf may be applied to the plant, or less than about 50 μg / ml, less than about 40, 30, 20, 10, or 5 μg / ml, less than about 1 μg / ml, or about 0.5 μg / ml of the recombinant RNA molecule / leaf may be applied to the plant. In some respects, recombinant RNA molecules can be applied to plants at concentrations of less than about 50 ng / ml, less than about 40, 30, 20, 10, or about 5 ng / ml, or less than about 1 ng / ml per plant or plant leaf. In some respects, the disclosed recombinant RNA molecules are applied to plants at concentrations of about 10 ng / ml.

[0193] In some aspects, contacting insect pests with the disclosed insecticidal composition includes applying a composition comprising recombinant RNA molecules (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA) to the surface of the insect pest or to the surface of a plant infested by the insect pest. In some aspects, contact leads to insect mortality (death) or developmental retardation (growth retardation, or slowing or cessation of metamorphosis), thereby preventing or treating plant infestation by the insect. In some aspects, contact induces physiological or behavioral changes in insects (adults, larvae, or nymphs) that reduce the insect's ability to infest or damage plants, such as reduced reproductive capacity, or reduced or ceased feeding behavior or activity. In some aspects, application of an effective amount of recombinant RNA to a crop plant improves the crop plant's yield (e.g., increased biomass, increased seed or fruit production, or increased oil, starch, sugar, or protein content) compared to untreated crop plants. In some aspects, feeding assays are used to determine the efficacy of the recombinant RNA molecule trigger in inducing developmental retardation or mortality in insect pests.

[0194] In some respects, contacting insecticidal compositions containing recombinant RNA molecules (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA) with insecticidal compositions results in at least about 10% mortality, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, compared to the mortality rates of insect pests before composition application, control insect pests that did not receive the composition, or insect pests that came into contact with recombinant RNA molecules containing non-insect pest targets (e.g., white spot syndrome virus genes). In some respects, contacting insect pests with insecticidal compositions described herein results in at least 25% insect pest mortality. Insect pest mortality can be evaluated using the feeding assays described in the examples.

[0195] In some respects, the insecticidal composition is applied to plant-infecting insects, wherein the insects are Coleoptera. In some respects, the insecticidal composition is applied to insect pests, wherein the insects are species of the genus *Lespedeza*. In some respects, the insects are *Potato beetle* (Colorado potato beetle), *False potato beetle*, or *Texas potato beetle* (Texas false potato beetle). In some respects, the insect is *Potato beetle* (Colorado potato beetle). In some respects, the insect is an adult, or in a larval or nymphal stage.

[0196] In some aspects, an insecticidal composition is applied to a plant-infecting insect, said insect being the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence that contains at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence that contains 100% identity with SEQ ID No: 1.

[0197] In some aspects, the insecticidal composition is applied to a plant-infecting insect, said insect being the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence containing 100% identity with SEQ ID No: 4.

[0198] In some aspects, the insecticidal composition is applied to a plant-infecting insect, said insect being the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence that contains at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence that contains 100% identity with SEQ ID No: 6.

[0199] In some aspects, the insecticidal composition is applied to a plant-infecting insect, said insect being the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence that contains at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence that contains 100% identity with SEQ ID No: 8.

[0200] In some aspects, the insecticidal composition is applied to a plant-infecting insect, said insect being the potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 3.

[0201] In some aspects, the insecticidal composition is applied to a plant-infecting insect, said insect being the potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 5.

[0202] In some aspects, the insecticidal composition is applied to a plant-infecting insect, said insect being the potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 10.

[0203] C. Usage Method This article further discloses methods for controlling insect pest infestation. In some aspects, the methods involve applying a composition containing recombinant RNA molecules (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA) to the surface of an insect or the surface of a plant infested by the insect.

[0204] In some aspects, this document discloses methods for inducing mortality or inhibiting the growth of insect pests, comprising contacting the insect pests with the insecticidal composition disclosed herein. In further aspects, methods for reducing the growth of insect pests on plants or reducing insect pest infestation on plants are disclosed, comprising applying an effective amount of the insecticidal composition disclosed herein to the plant. In some aspects, this document provides methods for improving plant yield, comprising applying an effective amount of the insecticidal composition to the plant.

[0205] In some aspects, methods for controlling plant infection by plant-infectious insects include contacting the insect pests with the recombinant RNA disclosed herein. In such aspects, the recombinant RNA comprises at least one portion having 17 or more adjacent nucleotides having a sequence that is about 95% to about 100% complementary to a portion of a target gene of the plant-infecting insect. In some aspects, the target gene is LdEG12, LdEG53, LdEG81, or any combination thereof. In some aspects, the target gene encodes an mRNA sequence selected from SEQ ID Nos: 1, 4, 6, and 8.

[0206] In some aspects, the disclosed methods involve contacting an insect pest with a recombinant RNA molecule comprising a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity with a portion of a target gene, wherein the target gene is LdEG12. In such aspects, the target gene encodes the mRNA sequence of SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence containing about 100% identity with the sequence of SEQ ID No: 1.

[0207] In some aspects, the disclosed methods include contacting an insect pest with a recombinant RNA molecule comprising a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity with a portion of a target gene, wherein the target gene is LdEG53. In such aspects, the target gene encodes an mRNA sequence of SEQ ID No: 4, SEQ ID No: 6, or any combination thereof. In some aspects, the target gene encodes an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence containing about 100% identity with the sequence of SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence containing about 100% identity with the sequence of SEQ ID No: 6.

[0208] In some aspects, the disclosed methods involve contacting an insect pest with a recombinant RNA molecule comprising a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity with a portion of a target gene, wherein the target gene is LdEG81. In such aspects, the target gene encodes the mRNA sequence of SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with the sequence of SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence containing about 100% identity with the sequence of SEQ ID No: 8.

[0209] In some aspects, the disclosed method includes contacting an insect pest with a recombinant RNA molecule containing at least one RNA strand, said at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID NO: 3, 5 and 10.

[0210] In some aspects of this method, the target gene is LdEG12, and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 3. In other aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 3. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 3.

[0211] In some aspects of this method, the target gene is LdEG53, and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 5. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 5. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 5.

[0212] In some aspects of this method, the target gene is LdEG81, and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with the sequence of SEQ ID NO: 10. In other aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with the sequence of SEQ ID NO: 10. In a further aspect, the recombinant RNA molecule comprises at least one RNA strand having a sequence having at least about 100% identity or complementarity with the sequence of SEQ ID NO: 10.

[0213] In some aspects of this method, the recombinant RNA molecule is dsRNA, miRNA, siRNA, hpRNA, or pi-RNA.

[0214] In some cases, the target gene is LdEG12, and the recombinant RNA molecule is dsRNA, miRNA, siRNA, hpRNA, or pi-RNA. In other cases, the target gene is LdEG53, and the recombinant RNA molecule is dsRNA, miRNA, siRNA, hpRNA, or pi-RNA. In still other cases, the target gene is LdEG81, and the recombinant RNA molecule is dsRNA, miRNA, siRNA, hpRNA, or pi-RNA.

[0215] In some aspects, the disclosed methods include contacting insect pests with dsRNA containing an RNA chain having a sequence selected from SEQ ID NO: 3, 5 and 10.

[0216] In some aspects, the target is LdEG12, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0217] In some aspects, the target is LdEG53, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0218] In some aspects, the target is LdEG81, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a dsRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0219] In some aspects, the target is LdEG12, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a miRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0220] In some aspects, the target is LdEG53, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a miRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0221] In some aspects, the target is LdEG81, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a miRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0222] In some aspects, the target is LdEG12, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a siRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0223] In some aspects, the target is LdEG53, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a siRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0224] In some aspects, the target is LdEG81, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a siRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0225] In some aspects, the target is LdEG12, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being an hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 3.

[0226] In some aspects, the target is LdEG53, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being an hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 5.

[0227] In some aspects, the target is LdEG81, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being an hpRNA comprising an RNA strand having a sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is an hpRNA comprising an RNA strand having a sequence having at least about 100% identity with SEQ ID NO: 10.

[0228] In some aspects, the target is LdEG12, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 3.

[0229] In some aspects, the target is LdEG53, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 5.

[0230] In some aspects, the target is LdEG81, and the method includes contacting an insect pest with a recombinant RNA molecule, said recombinant RNA molecule being a pi-RNA comprising an RNA strand having a complementary sequence having at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence having at least about 100% identity with SEQ ID NO: 10.

[0231] In some aspects, the method involves contact with plant-infecting insects, wherein said insects are Coleoptera. In some aspects, the method involves contact with insect pests, wherein said insects are species of the genus *Lespedeza*. In some aspects, the insect is the potato beetle (*Colonella potato beetle*), the false potato beetle (*Pseudopotato beetle*), or the Texas potato beetle (*Pseudopotato beetle*). In some aspects, the insect is the potato beetle (*Colonella potato beetle*). In some aspects, the insect is an adult, or in a larval or nymphal stage.

[0232] In some aspects, the method includes contacting an insecticidal composition with a plant-infectious insect, said insect being a potato beetle (Colorado potato beetle), and a target gene encoding an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence containing 100% identity with SEQ ID No: 1.

[0233] In some aspects, the method includes contacting an insecticidal composition with a plant-infectious insect, said insect being a potato beetle (Colorado potato beetle), and a target gene encoding an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence containing 100% identity with SEQ ID No: 4.

[0234] In some aspects, the method includes contacting an insecticidal composition with a plant-infectious insect, said insect being a potato beetle (Colorado potato beetle), and a target gene encoding an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence containing 100% identity with SEQ ID No: 6.

[0235] In some aspects, the method includes contacting an insecticidal composition with a plant-infectious insect, said insect being a potato beetle (Colorado potato beetle), and a target gene encoding an mRNA sequence containing at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity with SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence containing 100% identity with SEQ ID No: 8.

[0236] In some aspects, the method includes contacting an insecticidal composition with a plant-infectious insect, said insect being a potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprising at least one RNA strand having a sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 3. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 3.

[0237] In some aspects, the method includes contacting an insecticidal composition with a plant-infectious insect, said insect being a potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprising at least one RNA strand having a sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 5. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 5.

[0238] In some aspects, the method includes contacting an insecticidal composition with a plant-infectious insect, said insect being a potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprising at least one RNA strand having a sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 10. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 10.

[0239] In some aspects, the method involves contacting insect pests with a disclosed insecticidal composition containing recombinant RNA molecules (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA), or contacting a host cell expressing recombinant RNA (e.g., bacterial, fungal, or viral cells) with the surface of the insect pest or the surface of a plant infected by the insect pest. In some aspects of the method, contact results in insect mortality (death) or developmental retardation (growth retardation, or slowing or cessation of metamorphosis), thereby preventing or treating plant infection by the insect. In some aspects of the method, contact induces physiological or behavioral changes in the insect (adult, larva, or nymph) that reduce the insect's ability to infect or damage the plant, such as reduced reproductive capacity, or reduced or ceased feeding behavior or activity. In some aspects, application of an effective amount of recombinant RNA to a crop plant improves the crop plant's yield (e.g., increased biomass, increased seed or fruit production, or increased oil, starch, sugar, or protein content) compared to untreated crop plants. In some respects, feeding assays are used to determine the efficacy of recombinant RNA molecular triggers in causing developmental delays or mortality in insect pests.

[0240] In some aspects, methods involving contacting insect pests with an insecticidal composition containing a recombinant RNA molecule (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA) result in a mortality rate of at least about 10%, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, compared to the mortality rate of insect pests before application of the composition or as a control insect pest that has not received the composition. In some aspects, the methods involve contacting insect pests with the insecticidal composition described herein, resulting in a mortality rate of at least 25% of the insect pests. The mortality rate of the insect pests can be evaluated using any method known in the art, such as a feeding assay as described in the examples.

[0241] In another aspect, the methods disclosed herein can improve one or more plant properties, such as productivity, biomass, yield, or growth. For example, compared to the productivity, biomass, yield, or growth of plants that have not received an insecticidal composition of this disclosure, the methods disclosed herein can increase one or more of productivity, biomass, yield, and / or growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher.

[0242] In some respects, this document discloses methods for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding an RNA molecule containing a silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes the mRNA sequence of SEQ ID NO: 1. In such respects, the ingestion of RNA by the insect leads to mortality or developmental delay in the insect.

[0243] In some respects, this document discloses methods for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding an RNA molecule containing a silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes the mRNA sequence of SEQ ID NO: 4. In such respects, the ingestion of RNA by the insect leads to mortality or developmental delay in the insect.

[0244] In some respects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding an RNA molecule containing a silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes the mRNA sequence of SEQ ID NO: 6. In such respects, the ingestion of RNA by the insect leads to mortality or developmental delay in the insect.

[0245] In some respects, this document discloses methods for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding an RNA molecule containing a silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes the mRNA sequence of SEQ ID NO: 8. In such respects, the ingestion of RNA by the insect leads to mortality or developmental delay in the insect.

[0246] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding dsRNA, said dsRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 3. In some aspects, the dsRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 3. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0247] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding dsRNA, said dsRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 5. In some aspects, the dsRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 5. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0248] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding dsRNA, said dsRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 10. In some aspects, the dsRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 10. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0249] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding siRNA, said siRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 3. In some aspects, the siRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 3. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0250] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding siRNA, said siRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 5. In some aspects, the siRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 5. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0251] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding siRNA, said siRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 10. In some aspects, the siRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 10. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0252] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding a miRNA, said miRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 3. In some aspects, the miRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 3. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0253] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding a miRNA, said miRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 5. In some aspects, the miRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 5. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0254] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding a miRNA, said miRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 10. In some aspects, the miRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 10. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0255] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding hpRNA, said hpRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 3. In some aspects, the hpRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 3. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0256] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding hpRNA, said hpRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 5. In some aspects, the hpRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 5. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0257] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding hpRNA, said hpRNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 10. In some aspects, the hpRNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 10. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0258] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding pi-RNA, said pi-RNA comprising a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 3. In some aspects, the pi-RNA comprises at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 3. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0259] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding pi-RNA, said pi-RNA containing a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 5. In some aspects, the pi-RNA contains at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 5. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0260] In some aspects, this document discloses a method for providing plants with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding pi-RNA, said pi-RNA containing a nucleotide sequence having about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with SEQ ID NO: 10. In some aspects, the pi-RNA contains at least one RNA strand having a sequence having 100% identity or complementarity with SEQ ID NO: 10. In such aspects, the RNA expressed by the plant, when ingested by insect pests, leads to mortality or developmental delay in the insects.

[0261] In some aspects, methods of providing plants with improved resistance to infective insects include providing resistance to insect pests, said infective insects being adults, or in larval or nymphal stages. In some aspects, methods of providing plants with improved resistance to infective insects include providing resistance to insect pests, said infective insects being Coleoptera. In such aspects, infective insects are insects from the genus *Lepidocybe*. In some aspects, infective insects are the potato beetle (*Cotinus potato beetle*).

[0262] In some aspects, methods for expressing recombinant DNA constructs in plants can be employed by means of transgenic expression or transient expression. Transgenic plant cells and plants can be prepared using methods known in the art. In some aspects, the recombinant DNA constructs disclosed herein can be transcribed in any plant cell or tissue or in the whole plant at any developmental stage. Further provided are plants with improved resistance to insects, or fruits, seeds, or reproductive parts of plants with improved resistance, produced by the disclosed methods. In some aspects, commercial products derived from transgenic plant cells, plants, or seeds expressing recombinant DNA constructs conferring improved resistance are also disclosed herein. These commercial products include, but are not limited to, harvested leaves, roots, branches, tubers, stems, fruits, seeds, or other parts of plants; plant powders, oils, extracts, fermentation or digestion products, broken or whole grains or seeds; or any food or non-food products, including such commercial products derived from transgenic plant cells, plants, or seeds as disclosed herein.

[0263] In some respects, methods for providing plants with improved resistance to infective insects include providing at least about 10%, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, of resistance to insect pests compared to control plants that do not express the recombinant DNA construct. The plant's resistance to insect pests can be evaluated using the feeding assays described in the examples.

[0264] In some respects, transgenic plants express recombinant DNA constructs that confer resistance to insect pests (e.g., CPB), which are superimposed with other recombinant DNA constructs for conferring additional traits. Compared to plants lacking recombinant DNA constructs, the transgenic plants possess at least one additional modified trait selected from the following groups of traits: improved tolerance to abiotic stresses; improved tolerance to biotic stresses; modified primary metabolite composition; modified secondary metabolite composition; modified micronutrient, carotenoid, or vitamin composition; improved yield; improved ability to utilize nitrogen, phosphate, or other nutrients; modified agronomical characteristics; modified growth or reproductive characteristics; and improved harvest, storage, or processing quality.

[0265] In some respects, the methods disclosed herein can be further modified. As used herein, "modified method" can include modifying or altering one or more features or aspects of one or more steps of the disclosed method. In one aspect, the method can be modified, for example, by changing the amount of recombinant RNA molecules used in the disclosed method, or by changing the application frequency of the insecticidal composition in the disclosed method, changing the duration of contact between the insect pest and the insecticidal composition, or by replacing one or more of the disclosed components used in the composition with similar or equivalent components and / or reagents.

[0266] D. Reagent kit This document discloses a kit for treating insect infestations in plants, comprising at least one recombinant RNA molecule (e.g., dsRNA, miRNA, siRNA, hpRNA, or pi-RNA) as described herein, or a recombinant DNA construct, or a host cell, or a composition. The kit may be provided with suitable instructions for use. These instructions may be printed on suitable packaging in which other components are supplied, or may be provided as separate entities, such as leaflets or booklets. The instructions may be rolled or folded, for example, in the storage state, and then unfolded and flattened to guide the use of the remaining components of the kit. Example

[0267] The following embodiments illustrate specific aspects of the invention and its various uses. They are illustrative only and should not be considered as limiting the invention.

[0268] Methods and Materials Colorado potato beetle rearing conditions: Kennebec seed potatoes were purchased from Northshire Seed Farm (NorthDakota, USA) and grown in a greenhouse at 25°C for 4–6 weeks. Adult beetles were reared on whole Kennebec potato plants in netted Bugdorms (12x48 inches) at 75–100 adults per cage. These cages were maintained at 25–27°C with ~65–80% relative humidity. Depending on colony activity and oviposition, approximately 100 new adults were self-reared from eggs and added to the colony each month. For bioassays, eggs were collected from adult cages and placed in 48-well insect rearing trays at one clutch per hole, and the trays were maintained in incubators with a 16:8 hour L:D photoperiod, 25°C, and 75% RH. Newly hatched larvae were synchronized according to their pupal dates and transferred to fresh leaf food until they reached the second instar. Bioassays were performed using larvae in the early L2 stage, which was confirmed by the width of the head capsule (width: 1 mm).

[0269] dsRNA production for bioassays:RNAiSSANCE Ag's proprietary technology for dsRNA production in *E. coli* utilizes a 300-400 bp region from the coding sequence of each gene for dsRNA synthesis, following conventional in vitro transcription (IVT) methods or fermentation protocols. Detailed fermentation protocols are available in patent publication US 2022 / 0145294 A1. For both methods, the 300-400 bp DNA sequence is purchased from the vendor (gBlocks GeneFragments, Integrated DNA Technologies). For the IVT method, the DNA fragment is amplified by PCR using primers having a T7 promoter sequence attached to the primer or included in the gBlock gene fragment, such that the PCR product has T7 promoter sequences attached to both ends. This PCR product is used in an in vitro transcription (IVT) reaction using the Megascript T7 Transcription Kit (ThermoFisher Scientific catalog number: AMB13345) to synthesize dsRNA. The amount of dsRNA was measured using Nanodrop (NanoDrop 2000 / 2000c Spectrophotometer, ThermoScientific™). For quantification of dsRNA produced by fermentation, an agarose gel-based method was used.

[0270] Screening for lethal genes using leaf disc bioassays:The insecticidal efficacy of the CPB gene targets was evaluated using a laboratory leaf disc bioassay protocol to assess how well the orally delivered double-stranded RNA (dsRNA) performed its intended function of killing CPB larvae, as measured by corrected insect mortality recorded during the assay period. For the screening bioassay, the target dsRNA treatment was prepared at a concentration of 10 ng / µL. When two sequences from each gene were used in any assay, 10 ng / µL dsRNA from each gene was mixed as a treatment. Following a randomized fully block design, 20 early L2 stage larvae were used in the bioassay. Each larva was placed in each well of a white 32-well rearing tray (RT32W, Frontier Agricultural Sciences), with 4 insects forming a pseudo-repeat, and 5 such replicates were used in one assay. Each replicate (4 insects) was placed on a separate tray. A 50 mm diameter Whatman circular filter paper (Cytiva, catalog 1004-050) was placed in each well, and 300 µL of distilled water was added to the filter paper to provide moisture so that the leaf discs did not dry out. Using a leaf cutter, 2 cm diameter circular leaf discs (area = 3.14 cm²) were cut from fully flattened, detached leaves collected from 6–8 week old potato plants (potato 'Kennebec'). 24 µL of dsRNA solution was applied to the leaf discs, and the solution was spread using a plastic or glass spreader. Tween-20 (0.1%, v / v final concentration) was used as a surfactant. Once the leaf discs were completely dry, one disc was placed in each well, and a larva was placed inside the well using a fine brush. The larvae were starved for 5 hours prior to infection to encourage immediate feeding after placement inside the well. Finally, the wells were sealed with a transparent sealing tape (RTCV4, Frontier Agricultural Sciences). The bioassay trays were maintained in a standard incubator / growth chamber at 25°C with a 16:8-hour L:D photoperiod and 60-65% humidity. Following the above steps, on day 2 (after 48 hours), the leaf trays were replaced with new dsRNA-treated leaf trays. On day 4 (after 96 hours), the treated leaf trays were replaced with untreated leaf trays. The assays lasted approximately 8 days. On the last day of the assays, larval mortality was scored. Each assay was repeated twice.

[0271] Whole plant bioassay:Bioassays were performed using larvae in the early L2 stage. For whole-plant bioassays, 6-8 week old potato plants (Variegata Kennebec) were used. The lower leaves of the plants were trimmed to obtain plants of appropriate size. One plant was used as a biological replicate, and 10 mL of E. coli-encapsulated dsRNA was sprayed onto each plant using a handheld spray bottle until the leaves and stems dripped. The concentration of dsRNA in the E. coli cells was 52 ng / µL (52 ppm). The sprayed plants were allowed to stand at room temperature until completely dry. After a 5-hour starvation period, 10 early L2 stage CPB larvae were randomly placed on the plants. A total of 5 replicates (5 plants, 10 larvae per plant) were used for the target gene, with the same number of plants / insects sprayed with E. coli cells without dsRNA as an untreated control. After infection, the plants were placed inside a 12" x 20" circular butterfly cage (Restcloud, ASIN B07HMMGRR2). The total duration of the bioassay was 14 days, with leaf consumption measured by visual inspection on day 14.

[0272] Data Analysis: All data were analyzed in R. To detect differences in mean mortality rates between treatments, a binomial model was created, followed by a one-way ANOVA. Lethal concentration values ​​were calculated using R. Survival The package is created, which includes the creation of a probit model.

[0273] Example 1: Procedure for screening target genes Using screening bioassays ( Figure 1 A screening was conducted on 95 genes selected from the larvae of the Colorado potato beetle (CPB). The genes tested were selected from a broad range of essential gene functional classes, including but not limited to protein processing, export and degradation, RNA synthesis, surveillance and turnover, energy production, oxidative phosphorylation, and others. In the screening bioassays, each dsRNA was provided as a 120 µL (10 ng / µL) concentration on detached leaves / discs. Treatments with two dsRNAs from two different genes consisted of a mixture of 10 ng / µL dsRNAs each. An appropriate concentration of non-target dsRNA was used as a control. Determinations in which treatments with non-target dsRNAs (negative control) resulted in a mortality rate greater than 20% were discarded. Figure 1 Mortality rates on the last day of the assays are depicted as indicated, and pooled results from several assays are shown (negative control mortality data are not shown). Three targets, LdEG12, LdEG53, and LdEG81, were selected for further characterization using assays including whole-plant bioassays, detached leaf / leaf disc bioassays, and lethal concentration (LC) assays, based on screening bioassays.

[0274] Example 2: Characterization of the bioactivity of LdEG12 To characterize the bioactivity of RNAi-mediated LdEG12 inhibition, CPB larvae (L2) were placed in 12-ounce cups with a mesh lid for ventilation. Excised leaves were preserved by placing the petioles inside microcentrifuge tubes containing 0.5% agar. The preserved excised leaves were placed in the cups and treated with 120 µL of 10 ppm (10 ng / µL) LdEG12 dsRNA or, as a negative control, the non-target shrimp white spot syndrome virus gene VP28 dsRNA (SEQ ID NO: 11). Five L2 developing larvae were then placed in each cup. Each treatment had six biological replicates. The larvae were exposed to dsRNA treatment for four days, after which untreated excised leaves were added to the cups. Figure 2A The percentage of larval mortality is shown. One-way ANOVA was performed on data observed on day 8 of treatment. Analysis showed that LdEG12 had a significantly higher mean larval mortality rate compared to the negative control (F1,10 = 75.34, p < 0.001 indicated by ***). Figure 2A ).

[0275] Further bioassays were performed by placing CPB L2 larvae in 150 x 25 mm culture dishes. Excised leaves, preserved by placing the stalks inside microcentrifuge tubes containing 0.5% agar, were placed in the culture dishes. The leaves were treated with 120 µl of 10 ppm (10 ng / µL) dsRNA LdEG12 or, as a negative control, non-target dsRNA and non-target dsRNA shrimp white spot syndrome virus gene VP28 dsRNA (SEQ ID NO: 11). Five L2 larvae were placed in each culture dish. Each treatment had six biological replicates. Larvae were exposed to dsRNA for four days, after which untreated excised leaves were added to the culture dishes. One-way ANOVA was performed on the data observed on day 7, and the results showed that LdEG12 had a significantly higher mean larval mortality rate on day 7 compared to the negative control (F1,10 = 375.6, p < 0.001 indicated by ***). Figure 2B ).

[0276] Bioassays were performed using 32-well trays. A randomized block design was used, with one CPB L2 larva placed in each well of the 32-well bioassay tray, where each block contained four larvae in four wells (pseudo-replicates); a total of 20 larvae were used for each treatment group, thus providing five true biological replicates for each treatment. Leaves were treated with 24 µl of 10 ppm (10 ng / µL) dsRNA LdEG12 or non-target dsRNA as a negative control. Larvae were exposed to dsRNA for four days, and the experiment was repeated three times. One-way ANOVA was performed on the data at day 8, and the results showed that LdEG12 had a significantly higher mean larval mortality rate compared to the negative control (F1,4 = 80.81, p < 0.001 indicated by ***). Figure 2C ).

[0277] To determine whether insect mortality was indeed caused by gene silencing, bioassays were performed in 32-well trays following a randomized block design, exposing CPB L2 larvae to 24 µL of 10 ppm (10 ng / µL) dsRNALdEG12 or non-target dsRNA on leaves for 4 days. After 4 days, surviving larvae were collected for RNA extraction and cDNA synthesis. Total RNA was extracted using the standard Trizol method, and cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TM (Catalogue No.: 4368814). cDNA was used in quantitative PCR (qPCR) using a Bullseye TaqProbe qPCR 2x Mastermix-Multiplex (MidSci entry # BEQPCR-PM) on a Bio-Rad CFX Opus 96 cycler. Relative gene expression of LdEG12 was calculated by comparing LdEG12 expression between LdEG12 dsRNA or non-target dsRNA treatments, and expression was normalized across treatments using RPL18 and ARF1 as reference genes (primer and probe sequences for LdEG12 and RPL18 are provided in Table 4). A t-test was run between the control and LdEG12 treatments. Figure 2D As shown, LdEG12 expression was significantly reduced in larvae treated with dsRNA LdEG12.

[0278] Furthermore, the lethal concentration of LdEG12 dsRNA was determined. Twenty L2-stage CPB larvae were placed in a 32-well bioassay tray, one larva per well. Leaf discs with a diameter of 20 mm were treated with 0.001 ppm, 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, or 100 ppm of dsRNA LdEG12 / disc. Untreated leaf discs served as controls. Larvae were continuously exposed to dsRNA for 8 days by replacing the leaf discs daily with freshly prepared dsRNA. The results from the experiment are shown in Table 1. LC on day 8 50 The concentration was determined to be 0.313 ppm (ng / µL), and the LC value was [data missing] on day 8. 90 The concentration was determined to be 3.81 ppm. LC was calculated using the Ecotox R package. Concentrations were log10 transformed, and estimates were fitted to a 95% confidence interval using a probabilistic unit model. Treatment mortality values ​​were corrected for untreated control mortality using the Handerson-Tifton formula. Mortality was defined as the number of insects that failed to move after repeated light touching with a paintbrush.

[0279] Table 1: LC concentration of dsRNA LdEG12 Percentage of deaths Concentration (ppm) LCL UCL Day 6 50 0.593 0.279 1.32 90 7.83 3 44 Day 7 50 0.349 0.156 0.809 90 5.45 1.99 32.9 Day 8 50 0.313 0.162 0.615 90 3.81 1.67 14.9 Two additional subunits of the ATPase / synthase complex—the b subunit (LdEG70) and the d subunit (LdEG118)—were screened to evaluate whether all ATPase / synthase subunits are essential for CPB biology. Additionally, the gene functioning in mitochondria—mitochondrial processing peptidase subunit α (…)—was also screened. PMPCALdEG102 was compared with LdEG12 to see if any of these genes involved in mitochondrial function were similar to LdEG12 and required. PMPCA proteins are not part of the oxidative phosphorylation pathway; however, they are localized in the mitochondrial matrix and function there by cleaving a leader peptide from a core or precursor protein recently introduced into the mitochondria. Most mitochondrial proteins are encoded by the nucleus, therefore they must translocate to the mitochondria. For proper localization, these proteins carry leader peptides to guide them to the mitochondria, and once there, these leader peptides are cleaved by mitochondrial peptidase to allow the protein to function correctly. For the bioassays performed, dsRNA targeting LdEG12, LdEG118, LdEG70, and LdEG102 (SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29) was used in a 32-well tray assay, as described above. The top inset plot shows the percentage mortality of larvae treated with dsRNA separately on day 8, and the bottom inset plot shows the average leaf consumption as a percentage of leaf disc consumed by each larva on day 5. Data in both plots were analyzed using ANOVA and Fisher's LSD post-hoc test with a binomial model. Figure 2E As shown, targeting LdEG12 was the most effective at killing larvae compared to any of the other dsRNAs tested.

[0280] Table 2: CPB genes tested that are involved in mitochondrial function or are key components of the ATPase / synthase complex. Example 3: Characterization of the bioactivity of LdEG53 To characterize the bioactivity of RNAi-mediated LdEG53 inhibition, CPB larvae L2 were placed in 150 x 25 mm culture dishes. Excised leaves, preserved by placing the stalk inside a microcentrifuge tube containing 0.5% agar, were placed in the culture dishes. The leaves were treated with 120 µL of 10 ppm (10 ng / µL) dsRNA LdEG53 or, as a negative control, the non-target shrimp white spot syndrome virus gene VP28 dsRNA (SEQ ID NO: 11). Five L2 larvae were placed in each culture dish. On day 4, one live larva from each group was obtained for qPCR analysis. Each treatment was repeated six times. Larvae were exposed to dsRNA for 4 and 8 days respectively. Figure 3A Results from the experiment showed that, using one-way ANOVA, there were significant differences in mean larval mortality on day 8 (F1,6 = 18.44, p < 0.01 indicated by ***).

[0281] To determine whether insect mortality was indeed due to gene silencing, bioassays were performed on LdEG53 following the same protocol described above for LdEG12. The qPCR primer and probe sequences for LdEG53 are provided in Table 4. A t-test was run between the control and LdEG53 treatments. Figure 3B The results showed that LdEG53 expression was actually reduced in larvae exposed to dsRNA LdEG53.

[0282] Furthermore, using a method similar to that described above for LdEG12, the lethal concentration of dsRNA LdEG53 was identified. The LC concentration at day 8... 50 The concentration was determined to be 0.059 ppm or ng / µL, and the LC value was determined on day 8. 90 The concentration was identified as 0.501 ppm (Table 3).

[0283] Table 3: LC concentration of dsRNA LdEG53 In addition, LdEG53 dsRNA activity was tested in whole plants. 3–5 week old Kennebec potato plants were treated with 52 ppm of LdEG53 dsRNA. Untreated plants served as controls. 10 mL of dsRNA solution was applied to each plant once using a fine mist spray bottle (Fantasea, model #BX-FSC296). Ten L2 CPB larvae were placed on each plant inside a butterfly cage. Five cages / plants / treatments were used. Plants and larvae were maintained at room temperature, under greenhouse conditions, or at approximately 25°C in a growing tent. Mortality was recorded for up to 14 days. The experiment was repeated four times. Significant differences between experimental replicates were identified using a two-factor ANOVA (F-value). 1,3,35 = 4.009, p<0.05). A significant difference in mean larval mortality was observed between L2 CPB larvae placed on LdEG53-treated and untreated plants at day 14 (F<0.05). 1,3,35 =454.711, p<0.001 as indicated by ***) Figure 3C ).

[0284] Example 4: Characterization of the bioactivity of LdEG81 The activity of LdEG81 dsRNA was characterized by screening for lethality of LdEG81 dsRNA. One CPB larva was placed in each well of a 32-well bioassay tray. Leaves were treated with 24 µl of 10 ppm LdEG81 dsRNA or a non-target negative control dsRNA (shrimp white spot syndrome virus gene VP28 dsRNA, SEQ ID NO: 11). Each treatment included 20 insects. Larvae were exposed to either dsRNA or the non-target negative control dsRNA for 4 days, and the percentage mortality of CPB larvae was recorded on days 2, 4, 7, 8, and 10. The application of LdEG81 dsRNA showed a significantly greater mortality rate in CPB larvae compared to the non-target negative control dsRNA. Figure 4A ).

[0285] Furthermore, whole-plant assays were performed using LdEG81 dsRNA. Whole Kennebec potato plants aged 3–5 weeks were treated with 52 ppm LdEG81 dsRNA. Untreated plants were used as a control. Ten L2 CPB larvae were placed on the plants inside butterfly cages. Five cages / treatment were present. Plants and larvae were held in a growing tent or greenhouse at approximately 25°C and 60–65% relative humidity. Mortality was recorded for up to 14 days. LdEG81-treated plants showed a significantly higher larval mortality rate (F0.05) on day 14 compared to untreated plants. 1,28 = 69.82, p<0.001) Figure 4B ).

[0286] Implementation Examples Overview Three target genes, LdEG12, LdEG53, and LdEg81, were identified, and their lethality in CPB larvae was tested using downregulation of RNAi targeting the identified genes. Both in vitro and whole-plant assays showed that CPB larvae exposed to dsRNAs targeting LdEG12, LdEG53, or LdEg81 had higher mortality rates compared to control CPB. These results indicate that LdEG12, LdEG53, and LdEg81 are essential genes in CPB, and that downregulation of these genes using RNAi is suitable for insecticidal purposes.

[0287] Table 4: Sequences

Claims

1. An insecticidal composition comprising an insecticidally effective amount of a recombinant RNA molecule, said recombinant RNA molecule comprising a nucleotide sequence having at least 17 or more adjacent nucleotides having a sequence having at least about 95% to about 100% complementarity to a portion of a target gene of an insect infecting a plant, said target gene encoding an mRNA sequence selected from SEQ ID No: 1, 4, 6 and 8.

2. The insecticidal composition according to claim 1, wherein the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID NO: 3, 5 and 10.

3. The insecticidal composition according to claim 1, wherein the recombinant RNA molecule is double-stranded RNA (dsRNA), microRNA (miRNA), small interfering RNA (siRNA), hairpin RNA (hpRNA), or piwi-interacting RNA (pi-RNA).

4. The insecticidal composition according to claim 1, wherein the recombinant RNA molecule is a double-stranded RNA (dsRNA) comprising an RNA chain having a sequence selected from SEQ ID NO: 3, 5 and 10.

5. The insecticidal composition according to claim 1, wherein the dsRNA has a length of at least 17 base pairs.

6. The insecticidal composition according to claim 1, wherein the insect is an adult, or in the larval or nymphal stage.

7. The insecticidal composition according to claim 1, wherein the plant-infecting insect is a Coleoptera insect.

8. The insecticidal composition according to claim 7, wherein the plant-infecting insect is an insect from the genus *Lepidium*.

9. The insecticidal composition of claim 8, wherein the plant-infecting insect is the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No:

1.

10. The insecticidal composition of claim 1, wherein the plant-infecting insect is the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No: 4 or 6.

11. The insecticidal composition of claim 1, wherein the plant-infecting insect is the potato beetle (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No:

8.

12. The insecticidal composition of claim 1, wherein the plant-infecting insect is the potato beetle (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID No: 3, 5 and 10.

13. The insecticidal composition of claim 1, wherein the plant-infecting insect is the potato beetle (Colorado potato beetle), and the recombinant RNA molecule is dsRNA comprising an RNA chain having a sequence selected from SEQ ID No: 3, 5, and 10.

14. The insecticidal composition according to claim 1, further comprising at least one component selected from carriers, excipients, diluents, surfactants, organosilicones, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide pesticides, safeners, insect attractants, and insect growth regulators.

15. The insecticidal composition according to claim 1, further comprising at least one pesticide agent.

16. The insecticidal composition according to claim 14, wherein the pesticide reagent is selected from potato glycoprotein, phytohemagglutinin, phytoecdysone, and insecticidal protein.

17. The insecticidal composition according to claim 1, wherein it is in the form of: solid, liquid, powder, suspension, emulsion, spray, encapsulating agent, microbeads, carrier microparticles, film, solid matrix, soil irrigation agent, insect food, insect bait and seed treatment agent.

18. A plant or its seeds treated with the insecticidal composition according to claim 1, wherein the plant exhibits improved resistance to the insect.

19. A recombinant DNA construct comprising a heterologous promoter operatively linked to DNA encoding an RNA transcript, said RNA transcript comprising a sequence having about 95% to about 100% identity or complementarity with a sequence selected from SEQ ID NO: 3, 5 and 10.

20. The recombinant DNA construct of claim 19, wherein the heterologous promoter is functional for the expression of RNA transcripts in bacteria or fungi.

21. The recombinant DNA construct of claim 19, wherein the heterologous promoter is functional in plant cells.

22. A recombinant vector comprising the recombinant DNA construct according to claim 19.

23. A plant chromosome or plastid comprising the recombinant DNA construct according to claim 22.

24. A transgenic plant cell having in its genome the recombinant DNA construct according to claim 22.

25. A transgenic plant comprising the transgenic plant cells according to claim 24.

26. A crop product produced from a genetically modified plant according to claim 25.

27. The transgenic offspring seeds or fertile plant parts of the transgenic plant according to claim 25.

28. A method for preventing plant infection by an infectious insect, the method comprising contacting the insect with dsRNA, the dsRNA comprising at least one portion having 17 or more adjacent nucleotides having a sequence having about 95% to about 100% complementarity to a target gene of the insect or a portion thereof, wherein the target gene encodes an mRNA sequence selected from SEQ ID NO: 1, 4, 6 and 8.

29. The method of claim 28, wherein the insect is an adult, or in the larval or nymphal stage.

30. The method of claim 28, wherein the plant-infecting insect is a Coleoptera insect.

31. The method of claim 30, wherein the plant-infecting insect is an insect from the genus *Lepidium*.

32. The method of claim 31, wherein the insect is a potato beetle (Colorado potato beetle).

33. The method of claim 28, wherein the dsRNA comprises a sequence selected from SEQ ID No: 3, 5 and 10.

34. The method of claim 28, wherein the dsRNA comprises more than one portion having 17 or more adjacent nucleotides having a sequence that is about 95% to about 100% complementary to the target gene or a portion thereof.

35. The method of claim 28, wherein the dsRNA is blunt-ended.

36. The method of claim 28, wherein the dsRNA has a protruding end at at least one end.

37. The method of claim 28, wherein the dsRNA is (a) chemically synthesized, or (b) produced by expression in microorganisms, expression in plant cells, or by microbial fermentation.

38. The method of claim 28, wherein the dsRNA is chemically modified.

39. The method of claim 28, wherein the contact comprises applying a composition containing dsRNA to the surface of an insect or the surface of a plant infected by an insect.

40. The method of claim 28, wherein the composition comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulating agent, microbeads, carrier microparticles, film, matrix, or seed treatment agent.

41. The method of claim 28, wherein the contact comprises providing dsRNA in the composition, the composition further comprising one or more components selected from carrier reagents, surfactants, organosilicones, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide pesticides, safeners, insect attractants, and insect growth regulators.

42. The method of claim 28, wherein the contact comprises providing dsRNA in the composition, and the composition further comprises at least one pesticide agent.

43. The method according to claim 42, wherein the pesticide reagent is selected from potato glycoprotein, phytohemagglutinin, phytoecdysone, and insecticidal protein.

44. The method of claim 28, wherein the contact comprises providing dsRNA in the composition ingested by the insect.

45. The method of claim 44, wherein the ingested composition further comprises one or more components selected from carrier reagents, surfactants, organosilicones, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide pesticides, safeners, insect attractants, and insect growth regulators.

46. ​​A method of causing mortality in insects, comprising providing an insecticidal composition according to any one of claims 1-17, or a plant according to claim 18, to the insect's food, wherein, upon ingestion by the insect, the composition or plant causes mortality or stunted growth in the insect.

47. The method of claim 46, wherein the insect is an adult, or in the larval or nymphal stage.

48. A method of providing a plant with improved resistance to infective insects, the method comprising introducing into the plant a recombinant DNA construct expressing a nucleotide sequence encoding an RNA molecule containing a silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of an insect, wherein the target gene encodes an mRNA sequence selected from SEQ ID No: 1, 4, 6 and 8, and wherein ingestion of the RNA by the insect results in mortality or developmental delay in the insect.

49. The method of claim 42, wherein the silencing element has a sequence having about 95% to about 100% sequence identity or complementarity with a sequence selected from SEQ ID No: 3, 5 and 10.

50. The method of claim 42, wherein the recombinant DNA construct further comprises a heteropromoter operatively linked to a nucleotide sequence encoding an RNA molecule and functional in plant cells.

51. The method of claim 42, wherein the silencing element is dsRNA, miRNA, small siRNA, hpRNA, or pi-RNA.

52. The method of claim 42, wherein the plant-infectious insect is an adult, or in the larval or nymphal stage.

53. The method according to claim 42, wherein the plant-infecting insect is a Coleoptera insect.

54. The method of claim 42, wherein the plant-infecting insect is an insect from the genus *Lepidium*.

55. The method of claim 42, wherein the plant-infecting insect is the potato beetle (Colorado potato beetle).

56. The method of claim 42, wherein the introduction comprises transgenic expression or transient expression.

57. A plant produced by the method according to claim 42 and having improved resistance to said insect.

58. The fruit, seed, or fertile part of the plant according to claim 57.

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